MarA family helix-turn-helix domains and their methods of use

ABSTRACT

An important advance in the battle against drug resistance by elucidating the domains of MarA which are critical in mediating its function. Accordingly, MarA family protein helix-tun-helix domains, mutant MarA family protein helix-turn-helix domains and methods of their use, for example, in screening assays to identify compounds which are useful as antiinfective agents and in screening assays to identify loci which are involved in mediating antibiotic resistance are described.

RELATED APPLICATIONS

[0001] This application is a continuation application of U.S. patentapplication Ser. No. 09/316,504, filed May 21, 1999, which claimspriority to U.S. Ser. No. 60/086,497, filed on May 22, 1998. Thecontents of these applications are specifically incorporated herein byreference.

GOVERNMENT FUNDING

[0002] This work was funded, in part, by USPHS grant GM51661. Thegovernment may, therefore, have certain rights to this invention.

BACKGROUND OF THE INVENTION

[0003] Multidrug resistance in bacteria is generally attributed to theacquisition of multiple transposons and plasmids bearing geneticdeterminants for different mechanisms of resistance (Gold et al. 1996.N. Engl. J. Med. 335:1445). However, descriptions of intrinsicmechanisms that confer multidrug resistance have begun to emerge. Thefirst of these was a chromosomally encoded multiple antibioticresistance (mar) locus in Escherichia coli (George and Levy. 1983. J.Bacteriol. 155:531; George and Levy 1983. J. Bacteriol. 155:541). Marmutants of E. coli arose at a frequency of 10⁻⁶ to 10⁻⁷ and wereselected by growth on subinhibitory levels of tetracycline orchloramphenicol (George and Levy, supra). These mutants exhibitedresistance to tetracyclines, chloramphenicol, penicillins,cephalosporins, puromycin, nalidixic acid, and rifampin (George andLevy, supra). Later, the resistance phenotype was extended to includefluoroquinolones (Cohen et al. 1989. Antimicrob. Agents Chemother.33:1318), oxidative stress agents (Ariza et al. 1994. J. Bacteriol.176:143; Greenberg et al. 1991. J. Bacteriol. 173:4433), and morerecently, organic solvents (White et al. 1997. J. of Bacteriology179:6122; Asako, et al. 1997. J. Bacteriol. 176:143) and householddisinfectants, e.g., pine oil and/or triclosan® (McMurry et al. 1998.FEMS Microbiology Letters 166:305; Moken et al. 1997. AntimicrobialAgents and Chemotherapy 41:2770).

[0004] The expression of the Mar phenotype is greater at 30° C. than at37° C. (Seoane and Levy. 1995. J. Bacteriol. 177:3414). Continued growthin the same or higher antibiotic concentrations led to increased levelsof resistance, thus demonstrating a multiple antibiotic resistancephenotype which could be amplified (George and Levy, supra). Both high-and low-level resistance were decreased or completely reversed by a Tn5insertion into a single locus at 34 min (1,636.7 kb) on the E. colichromosome, called the mar locus. The genetic basis for high-levelresistance is only partially attributed to the mar locus, sincetransduction of the locus from high-or low-level mar mutants producesonly a low level of multidrug resistance.

[0005] The mar locus consists of two divergently positionedtranscriptional units that flank a common promoter/operator region in E.coli and Salmonella typhimurium (Alekshun and Levy. 1997. AntimicrobialAgents and Chemother. 41: 2067). One operon encodes MarC, a putativeintegral inner membrane protein without any yet apparent function, butwhich appears to contribute to the Mar phenotype in some strains. Theother operon comprises marRAB, encoding the Mar repressor (MarR), whichbinds marO and negatively regulates expression of marRAB (Cohen et al.1994. J. Bacteriol. 175:1484; Martin and Rosner. 1995. Proc. Natl. Acad.Sci. USA 92:5456; Seoane and Levy. 1995. J. Bacteriol. 177:530), anactivator (MarA), which controls expression of other genes on thechromosome, e.g., the mar regulon (Cohen et al. 1994. J. Bacteriol.175:1484; Gambino et. al. 1993. J. Bacteriol. 175:2888; Seoane and Levy.1995. J. Bacteriol. 177:530), and a putative small protein (MarB) ofunknown function.

[0006] MarA is a member of the XylS/AraC family of transcriptionalactivators (Gallegos et al. 1993. Nucleic Acids Res. 21:807). Proteinswithin this family activate many different genes, some of which produceantibiotic and oxidative stress resistance or control microbialmetabolism and virulence (Gallegos et al. supra).

SUMMARY

[0007] The present invention represents an important advance in thebattle against drug resistance by elucidating the domains of MarA whichare critical in mediating its function. Accordingly, the inventionprovides, inter alia, MarA family protein helix-turn-helix (HTH)domains, mutant MarA family protein helix-turn-helix domains and methodsof their use. This new understanding of how MarA family proteins work toactivate gene transcription will be invaluable to understanding andultimately controlling multidrug resistance.

[0008] In one aspect, the invention pertains to a method for identifyingan antiinfective compound which affects the activity of a MarA familyhelix-turn-helix domain, by contacting a polypeptide comprising a MarAfamily helix-turn-helix domain derived from a MarA family protein with acompound under conditions which allow interaction of the compound withthe polypeptide such that a complex is formed; and measuring the abilityof the compound to affect the activity of a MarA family helix-turn-helixdomain as an indication of whether the compound is an antiinfectivecompound.

[0009] In another aspect, the invention pertains to a method foridentifying an antiinfective compound which affects the activity of aMarA family helix-turn-helix domain, by contacting a cell expressing aMar A family helix-turn-helix domain polypeptide derived from a MarAfamily protein with a compound under conditions which allow interactionof the compound with the polypeptide; and measuring the ability of thecompound to affect the activity of a MarA family helix-turn-helix domainpolypeptide as an indication of whether the compound is an antiinfectivecompound.

[0010] In one embodiment, the step of measuring the ability of thecompound to affect the activity of a MarA family helix-turn-helix domaincomprises detecting the ability of the complex to activate transcriptionfrom a MarA family member responsive promoter. In a preferredembodiment, the Mar A responsive promoter is selected from the groupconsisting of marO, micF, and fumC

[0011] In one embodiment, the Mar A responsive promoter is linked to areporter gene. In a preferred embodiment, the reporter gene is selectedfrom the group consisting of lacZ, phoA, or green fluorescence protein.

[0012] In one embodiment, the step of measuring comprises measuring theamount of reporter gene product. In another embodiment, the step ofmeasuring comprises measuring the amount of RNA produced by the cell. Inyet another embodiment, the step of measuring comprises measuring theamount of a protein produced by the cell. In still another embodiment,the step of measuring comprises using an antibody against a proteinproduced by the cell.

[0013] In another aspect, the invention pertains to a method foridentifying an antiinfective compound which affects the activity of aMarA family helix-turn-helix domain, by contacting a polypeptidecomprising a Mar A family helix-turn-helix domain derived from a MarAfamily protein with a compound in a cell-free system under conditionswhich allow interaction of the compound with the polypeptide such that acomplex is formed; and measuring the ability of the compound to affectthe activity of a MarA family helix-turn-helix domain as an indicationof whether the compound is an antiinfective compound.

[0014] In one embodiment, the MarA family helix-turn-helix domain is anisolated polypeptide and the step of measuring the ability of thecompound to affect the activity of a MarA family helix-turn-helix domaincomprises measuring the ability of the complex to bind to DNA.

[0015] In another embodiment of the invention, the method comprisesscreening a library of bacteriophage displaying on their surface a MarAfamily helix-turn-helix domain polypeptide, said polypeptide sequencebeing encoded by a nucleic acid contained within the bacteriophage, forability to bind a compound to obtain those compounds having affinity forthe helix-turn-helix domain, said method by contacting the phage whichdisplay the helix-turn-helix domain with a sample of a library ofcompounds so that the helix-turn-helix domain can interact with and forma complex with any compound having an affinity for the helix-turn-helixdomain; contacting the complex of the helix-turn-helix domain and boundcompound with an agent that dissociates the bacteriophage from thecompound; and identifying the compounds that bound to thehelix-turn-helix domain.

[0016] In another aspect, the invention pertains to a method forscreening a library of bacteriophage displaying on their surface aplurality of polypeptide sequences, each polypeptide sequence beingencoded by a nucleic acid contained within the bacteriophage, forability to bind an immobilized MarA family helix-turn-helix domain, toobtain those polypeptides having affinity for the helix-turn-helixdomain, said method by contacting the immobilized helix-turn-helixdomain with a sample of the library of bacteriophage so that thehelix-turn-helix domain can interact with the different polypeptidesequences and bind those having affinity for the helix-turn-helix domainto form a set of complexes consisting of immobilized helix-turn-helixdomain and bound bacteriophage; separating the complexes frombacteriophage which have not formed the complex; contacting thecomplexes of the helix-turn-helix domain and bound bacteriophage with anagent that dissociates the bound bacteriophage from the complexes; andisolating the dissociated bacteriophage and obtaining the sequence ofthe nucleic acid encoding the displayed polypeptide, so that amino acidsequences of displayed polypeptides with affinity for helix-turn-helixdomain are obtained.

[0017] In certain embodiments, the polypeptides of the inventioncomprise the helix-turn-helix domain most proximal to the carboxyterminus of the MarA family protein from which it is derived. In otherembodiments, the polypeptides of the invention comprise thehelix-turn-helix domain most proximal to the amino terminus of the MarAfamily protein from which it is derived. In preferred embodiments, thepolypeptides consist of the helix-turn-helix domain most proximal to thecarboxy terminus of the MarA family protein from which it is derived. Instill other preferred embodiments, the polypeptides consist of thehelix-turn-helix domain most proximal to the amino terminus of the MarAfamily protein from which it is derived.

[0018] In preferred embodiments of the invention, the MarA familyhelix-turn-helix domain is derived from a protein selected from thegroup consisting of: MarA, RamA, AarP, Rob, SoxS, and PqrA.

[0019] In certain embodiments of the invention, a compound identifiedusing the subject methods increases antibiotic susceptibility. In otherembodiments, a compound identified using the subject methods reducesinfectivity or virulenc of a microbe.

[0020] In certain embodiments of the invention, the compound iseffective against Gram negative bacteria. In other embodiments, thecompound is effective against Gram positive bacteria. In preferredembodiments, the Gram positive bacteria are from a genus selected fromthe group consisting of: Enterococcus, Staphylococcus, Clostridium andStreptococcus. In other preferred embodiments, the compound is effectiveagainst bacteria from the family Enterobacteriaceae. In still otherpreferred embodiments, the compound is effective against a bacteria of agenus selected from the group consisting of: Escherichia, Proteus,Klebsiella, Providencia, Enterobacter, Burkholderia, Pseudomonas,Aeromonas, Acinetobacter, and Mycobacteria.

[0021] In another aspect, the invention pertains to a cell based methodof identifying genetic loci in an microbe which affect antibioticresistance comprising introducing into said microbe a nucleotidesequence encoding a helix-turn-helix motif of a MarA family protein andassaying for changes in the antibiotic resistance profile of saidmicrobe. In certain embodiments, the invention further comprisesassaying for changes in transcription of genetic loci of said microbe.In other embodiments the invention further comprises identifyingproteins which are present in different amounts in resistant andsusceptible microbes. In other embodiments, the invention furthercomprising identifying the genes which encode said proteins.

[0022] In certain embodiments of the invention the antibiotic to whichsensitivity is measured is selected from the group consisting of:tetracycline, fluoroquinolones, chloramphenicol, penicillins,cephalosporins, puromycin, nalidixic acid, and rifampin.

[0023] In other embodiments, the antibiotic is a disinfectant,antiseptic, or surface delivered antibacterial compound. In yet otherembodiments, the antibiotic is an antifingal. In still otherembodiments, the antibiotic is an antiparasitic.

[0024] In another aspect, the invention pertains to a cell-free methodof identifying genetic loci in an microbe which affect resistance toantibiotics comprising contacting a nucleic acid molecule of saidmicrobe with a MarA family protein helix-turn-helix domain and allowingcomplexes to form; separating the nucleic acid molecule which has formeda complex with a helix-turn-helix domain from the helix-turn-helixdomain; and identifying the sequence of those nucleic acid moleculeswhich can bind to a MarA family protein helix-turn-helix domain.

[0025] In certain embodiments of the invention, the compound to betested is derived from a library of small molecules. In otherembodiments, the compound is a nucleic acid molecule. In still otherembodiments, the compound is an antisense or sense oligonucleitide. Inyet other embodiments, the compound is a naturally occurring smallorganic molecule.

[0026] In another aspect, the invention pertains to a kit foridentifying genetic loci in an microbe which affect resistance tocompounds comprising a nucleotide sequence encoding a naturallyoccurring helix-turn-helix domain of a MarA family protein and mutant,inactive form of a MarA family protein helix-turn-helix domain.

BRIEF DESCRIPTION OF THE DRAWINGS

[0027]FIG. 1 is a schematic showing how the MarA mutants of the presentinvention were constructed. Panel A shows the nucleotide sequence of themutagenic oligonucleotide. Panel B shows the wild-type MarA amino acidresidues 27 to 44, of MarA (based on the sequence provided in Cohen etal. 1993. J. Bacteriol. 175:1484). Mutants contained the amino acidsshown at the indicated sites of insertion in helix A and helix B of thefirst helix-turn-helix domain (i.e., the most amino terminalhelix-turn-helix domain) of MarA. Mutants differ in amino acidcomposition due to the mutagenic oligonucleotide inserted in oppositeorientations.

[0028]FIGS. 2A-2D shows an exemplary alignment of amino acid sequencesof selected MarA family protein family members; amino acid sequencesthat correspond to amino acids 30-76 of MarA are shown in panels A-B andamino acid sequences that correspond to amino acids 77-106 are shown inpanels C-D.

[0029] FIGS. 3A-B shows an alignment of amino acid sequences ofexemplary MarA family protein family members and MarA familyhelix-turn-helix domain consensus sequences.

[0030]FIG. 4 shows exemplary mutagenic oligomers for making mutations inthe second helix-turn-helix domain of MarA.

DETAILED DESCRIPTION

[0031] The present invention provides an advance in combating drugresistance by identifying the domains of MarA protein family memberswhich mediate resistance, methods of using these domains in drugscreening assays to identify compounds which interfere with themechanism of action of these domains, and methods of identifying othergenetic loci which are important in mediating antibiotic resistance invarious unrelated bacteria.

[0032] Before further description of the invention, certain termsemployed in the specification, examples and appended claims are, forconvenience, collected here.

[0033] I. Definitions

[0034] As used herein, the language “antiinfective compound” includes acompound which reduces the ability of a microbe to produce infection ina host. Antiinfective compounds include those compounds which are staticor cidal for microbes, e.g., an antimicrobial compound which inhibitsthe proliferation and/or viability of a microbe. Preferred antiinfectivecompounds increase the susceptibility of microbes to antibiotics ordecrease the infectivity or virulence of a microbe. The term “microbe”includes any unicellular microbe, e.g., bacteria, fungi, or protozoa.Therefore, agents which inhibit the proliferation and/or viability offungi or protozoa are also included in this term. In preferredembodiments, microbes are pathogenic for humans, animals, or plants,however in other embodiments, microbes are involved, e.g., in fouling orspoilage.

[0035] As used herein, the term “antibiotic” includes antimicrobialagents isolated from natural sources or chemically synthesized. The term“antibiotic” includes the antimicrobial agents to which the Marphenotype has been shown to mediate resistance and, as such, includesdisinfectants, antiseptics, and surface delivered compounds. Forexample, any antibiotic, biocide, or other type of antibacterialcompound, including agents which induce oxidative stress agents, andorganic solvents are included in this term. Preferred antibioticsinclude: tetracycline, fluoroquinolones, chloramphenicol, penicillins,cephalosporins, puromycin, nalidixic acid, and rifampin.

[0036] As used herein, the language “MarA family protein” includes themany naturally occurring transcription regulation proteins which havesequence similarities to MarA and which contain the MarA familysignature pattern, which can also be referred to as an XylS/AraCsignature pattern. An exemplary signature pattern which defines MarAfamily proteins is shown, e.g., on PROSITE and is represented by thesequence:[KRQ]-[LIVMA]-X(2)-[GSTALIV]-{FYWPGDN}X(2)-[LIVMSA]-X(4,9)-[LIVMF]-X(2)-[LIVMSTA]-X(2)-[GSTACIL]-X(3)-[GANQRF]-[LIVMFY]-X(4,5)-[LFY]-X(3)-[FYIVA]-{FYWHCM}-X(3)-[GSADENQKR]-X-[NSTAPKL]-[PARL],where X is any amino acid (SEQ ID NO:215). MarA family proteins have two“helix-turn-helix” domains. This signature pattern was derived from theregion that follows the first, most amino terminal, helix-turn-helixdomain (HTH1) and includes the totality of the second, most carboxyterminal helix-turn-helix domain (HTH2). (See the publicly availabledatabase PROSITE PS00041).

[0037] MarA family polypeptide sequences are “structurally related” toone or more known MarA family members, preferably to MarA. Thisstructural relatedness can be shown by sequence similarity between twoMarA family polypeptide sequences or between two MarA family nucleotidesequences. Sequence similarity can be shown, e.g., by optimally aligningMarA family member sequences using an alignment program for purposes ofcomparison and comparing corresponding positions. To determine thedegree of similarity between sequences, they will be aligned for optimalcomparison purposes (e.g., gaps may be introduced in the sequence of oneprotein for nucleic acid molecule for optimal alignment with the otherprotein or nucleic acid molecules). The amino acid residues or bases andcorresponding amino acid positions or bases are then compared. When aposition in one sequence is occupied by the same amino acid residue orby the same base as the corresponding position in the other sequence,then the molecules are identical at that position. If amino acidresidues are not identical, they may be similar. As used herein, anamino acid residue is “similar” to another amino acid residue if the twoamino acid residues are members of the same family of residues havingsimilar side chains. Families of amino acid residues having similar sidechains have been defined in the art (see, for example, Altschul et al.1990. J. Mol. Biol. 215:403) including basic side chains (e.g., lysine,arginine, histidine), acidic side chains (e.g., aspartic acid, glutamicacid), uncharged polar side chains (e.g., glycine, asparagine,glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains(e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine,methionine, tryptophan), beta-branched side chains (e.g., threonine,valine, isoleucine) and aromatic side chains (e.g., tyrosine,phenylalanine, tryptophan). The degree (percentage) of similaritybetween sequences, therefore, is a function of the number of identicalor similar positions shared by two sequences (i.e., % homology=# ofidentical or similar positions/total # of positions×100). Alignmentstrategies are well known in the art; see, for example, Altschul et al.supra for optimal sequence alignment.

[0038] MarA family polypeptides share some amino acid sequencesimilarity with MarA. The nucleic acid and amino acid sequences of MarAas well as other MarA family polypeptides are available in the art. Forexample, the nucleic acid and amino acid sequence of MarA can be found,e.g., on GeneBank (accession number M96235 or in Cohen et al. 1993. J.Bacteriol. 175:1484, or in SEQ ID NO:1 and SEQ ID NO:2).

[0039] The nucleic acid and/or amino acid sequences of MarA can be usedas “query sequences” to perform a search against databases (e.g., eitherpublic or private) to, for example, identify other MarA family membershaving related sequences. Such searches can be performed, e.g., usingthe NBLAST and XBLAST programs (version 2.0) of Altschul, et al. (1990)J. Mol. Biol. 215:403-10. BLAST nucleotide searches can be performedwith the NBLAST program, score=100, wordlength=12 to obtain nucleotidesequences homologous to MarA family nucleic acid molecules. BLASTprotein searches can be performed with the XBLAST program, score=50,wordlength=3 to obtain amino acid sequences homologous to MarA proteinmolecules of the invention. To obtain gapped alignments for comparisonpurposes, Gapped BLAST can be utilized as described in Altschul et al.,(1997) Nucleic Acids Res. 25(17):3389-3402. When utilizing BLAST andGapped BLAST programs, the default parameters of the respective programs(e.g., XBLAST and NBLAST) can be used. See http://www.ncbi.nlm.nih.gov.

[0040] MarA family members can also be identified as being structurallysimiliar based on their ability to specifically hybridize to nucleicacid sequences specifying MarA. Such stringent conditions are known tothose skilled in the art and can be found e.g., in Current Protocols inMolecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6. Apreferred, non-limiting example of stringent hybridization conditionsare hybridization in 6× sodium chloride/sodium citrate (SSC) at about45° C., followed by one or more washes in 0.2×SSC, 0.1% SDS at 50-65° C.Conditions for hybridizations are largely dependent on the meltingtemperature Tm that is observed for half of the molecules of asubstantially pure population of a double-stranded nucleic acid. Tm isthe temperature in ° C. at which half the molecules of a given sequenceare melted or single-stranded. For nucleic acids of sequence 11 to 23bases, the Tm can be estimated in degrees C. as 2(number of A+Tresidues)+4(number of C+G residues). Hybridization or annealing ofnucleic acid molecules should be conducted at a temperature lower thanthe Tm, e.g., 15° C., 20° C., 25° C. or 30° C. lower than the Tm. Theeffect of salt concentration (in M of NaCl) can also be calculated, seefor example, Brown, A., “Hybridization” pp. 503-506, in The Encyclopediaof Molec. Biol., J. Kendrew, Ed., Blackwell, Oxford (1994).

[0041] Preferably, the nucleic acid sequence of a MarA family memberidentified in this way is at least about 10%, 20%, more preferably atleast about 30%, more preferably at least about 40% identical and mostpreferably at least about 50%, or 60% identical or more with a MarAnucleotide sequence. Preferably, MarA family members have an amino acidsequence at least about 20%, more preferably at least about 30%, morepreferably at least about 40% identical and most preferably at leastabout 50%, or 60% or more identical with a MarA amino acid sequence.However, it will be understood that the level of sequence similarityamong microbial regulators of gene transcription, even though members ofthe same family, is not necessarily high. This is particularly true inthe case of divergent genomes where the level of sequence identity maybe low, e.g., less than 20% (e.g., B. burgdorferi as compared e.g., toB. subtilis). Accordingly, structural similarity among MarA familymembers can also be determined based on “three-dimensionalcorrespondence” of amino acid residues. As used herein, the language“three-dimensional correspondence” is meant to includes residues whichspatially correspond, e.g., are in the same functional position of aMarA family protein member as determined, e.g., by x-raycrystallography, but which may not correspond when aligned using alinear alignment program. The language “three-dimensionalcorrespondence” also includes residues which perform the same function,e.g., bind to DNA or bind the same cofactor, as determined, e.g., bymutational analysis.

[0042] Exemplary MarA family proteins are shown in Table 1, in FIGS. 2and 3, and at Prosite (PS00041) and include: AarP, Ada, AdaA, AdiY,AfrR, AggR, AppY, AraC, CafR, CelD, CfaD, CsvR, D90812, EnvY, ExsA,FapR, HrpB, InF, InvF, LcrF, LumQ, MarA, MelR, MixE, MmsR, MsmR, OrfR,Orf_f375, PchR, PerA, PocR, PqrA, RafR, RamA, RhaR, RhaS, Rns, Rob,SoxS, S52856, TetD, TcpN, ThcR, TmbS, U73857, U34257, U21191, UreR,VirF, XylR, XylS, Xys1, 2, 3, 4, Ya52, YbbB, YfiF, YisR, YzbC, and YijO.

[0043] In preferred embodiments, a MarA family protein excludes one ormore of XylS, AraC, and MelR. In other preferred embodiments, a MarAfamily protein is involved in antibiotic resistance. In particularlypreferred embodiments, a MarA family protein is selected from the groupconsisting of: MarA, RamA, AarP, Rob, SoxS, and PqrA.

[0044] Preferred MarA family polypeptides are “naturally occurring.” Asused herein, a “naturally-occurring” molecule refers to an MarA familymolecule having a nucleotide sequence that occurs in nature (e.g.,encodes a natural MarA family protein). In addition, naturally ornon-naturally occurring variants of these polypeptides and nucleic acidmolecules which retain the same functional activity, e.g., the abilityto bind to DNA and regulate transcription. Such variants can be made,e.g., by mutation using techniques which are known in the art.Alternatively, variants can be chemically synthesized. For example, itwill be understood that the MarA family polypeptides described herein,are also meant to include equivalents thereof. Such variants can bemade, e.g., by mutation using techniques which are known in the art.Alternatively, variants can be chemically synthesized. For instance,mutant forms of MarA family polypeptides which are functionallyequivalent, (e.g., have the ability to bind to DNA and to regulatetranscription from an operon) can be made using techniques which arewell known in the art. Mutations can include, e.g., at least one of adiscrete point mutation which can give rise to a substitution, or by atleast one deletion or insertion. For example, random mutagenesis can beused. Mutations can be made by random mutagenesis or using cassettemutagenesis. For the former, the entire coding region of a molecule ismutagenized by one of several methods (chemical, PCR, dopedoligonucleotide synthesis) and that collection of randomly mutatedmolecules is subjected to selection or screening procedures. In thelatter, discrete regions of a protein, corresponding either to definedstructural or functional determinants (e.g., the first or second helixof a helix-turn-helix domain) are subjected to saturating or semi-randommutagenesis and these mutagenized cassettes are re-introduced into thecontext of the otherwise wild type allele. In one embodiment, PCRmutagenesis can be used. For example, Megaprimer PCR can be used (O. H.Landt, Gene 96:125-128).

[0045] In certain embodiments, such variants have at least 60% aminoacid identity with a naturally occurring MarA family member protein. Inpreferred embodiments, such variants have at least about 70% amino acididentity with a naturally occurring MarA family member protein. In morepreferred embodiments, such variants have at least about 80% amino acididentity with a naturally occurring MarA family member protein. Inparticularly preferred embodiments, such variants have at least about90% amino acid identity and preferably at least about 95% amino acididentity with a naturally occurring MarA family member protein. In yetother embodiments, a nucleic acid molecule encoding a variant of a MarAfamily protein is capable of hybridizing under stringent conditions to anucleic molecule encoding a naturally occurring MarA family protein.

[0046] The language “mutant form of a MarA family helix-turn-helixdomain” includes mutant forms of such MarA family helix-turn-helixdomains which do not retain the same biological activity as thenaturally occurring form. For example, such mutants may not bind to aMarA family member promoter or may not initiate transcription from MarAfamily member responsive promoter or may initiate transcription at alower level than the naturally occurring MarA family member.

[0047] As used herein the language “activity of a MarA familyhelix-turn-helix domain” includes the ability of the helix-turn-helixdomain to interact with DNA, e.g., to bind to a MarA family proteinresponsive promoter, or to initiate transcription from such a promoter.

[0048] As used herein, the language “marA family protein responsivepromoter” includes promoters which initiate transcription of an operonin a microbe and is structurally or functionally related to the marApromoter, e.g., is bound by MarA or a protein related to MarA.Preferably, the marA family protein responsive promoter is a marRABpromoter. For example, in the mar operon, several promoters are marAfamily protein responsive promoters as defined herein, e.g., the 405-bpThaI fragment from the marO region is a marA family responsive promoter(Cohen et al. 1993. J. Bact. 175:7856). In addition, MarA has been shownto bind to a 16 bp MarA binding site (referred to as the “marbox” withinmarO (Martin et al. 1996. J. Bacteriol. 178:2216). MarA also initiatestranscription from the acrAB; micF; mlr 1,2,3; slp; nfo; inaA; fpr;sodA; soi-17,19; zwf; fumC; or rpsF promoters (Alekshun and Levy. 1997.Antimicrobial Agents and Chemother. 41:2067). Other marA familyresponsive promoters are known in the art and include: araBAD, araE,araFGH and araC, which are activated by AraC; Pm, which is activated byXylS; melAB which is activated by MelR; and oriC which is bound by Rob.

[0049] The language “MarA family protein responsive promoter” alsoincludes portions of the above promoters which are sufficient toactivate transcription upon interaction with a MarA family memberprotein. The portions of any of the MarA family protein-responsivepromoters which are minimally required for their activity can be easilydetermined by one of ordinary skill in the art, e.g, using mutagenesis.Exemplary techniques are described by Gallegos et al. (1996. J.Bacteriol. 178:6427). A “MarA family protein responsive promoter” alsoincludes non-naturally occurring homologs of MarA family proteinresponsive promoters which have the same function as naturally occurringMarA family promoters. Preferably such variants have at least 60%nucleotide sequence identity with a naturally occurring MarA familyprotein responsive promoter. In preferred embodiments, such variantshave at least about 70% nucleotide sequence identity with a naturallyoccurring MarA family protein responsive promoter. In more preferredembodiments, such variants have at least about 80% nucleotide sequenceidentity with a naturally occurring MarA family protein responsivepromoter. In particularly preferred embodiments, such variants have atleast about 90% nucleotide sequence identity and preferably at leastabout 95% nucleotide sequence identity with a naturally occurring MarAfamily protein responsive promoter. In yet other embodiments nucleicacid molecules encoding variants of MarA family protein responsivepromoters are capable of hybridizing under stringent conditions tonucleic acid molecules encoding naturally occurring MarA family proteinresponsive promoters.

[0050] The term “interact” includes close contact between molecules thatresults in a measurable effect, e.g., the binding of one molecule withanother. For example, a MarA family polypeptide can interact with a MarAfamily protein responsive promoter and alter the level of transcriptionof DNA. Likewise, compounds can interact with a MarA family polypeptideand alter the activity of a MarA family polypeptide.

[0051] As used herein, the term “multiple drug resistance (MDR)”includes resistance to both antibiotic and non-antibiotic compounds. MDRresults from the increased transcription of a chromosomal or plasmidencoded genetic locus in an organism, e.g., a marRAB locus, that resultsin the ability of the organism to minimize the toxic effects of acompound to which it has been exposed, as well as to other non-relatedcompounds, e.g., by stimulating an efflux pump(s) or microbiologicalcatabolic or metabolic processes.

[0052] As used herein the term “reporter gene” includes any gene whichencodes an easily detectable product which is operably linked to aregulatory sequence, e.g., to a MarA family protein responsive promoter.By operably linked it is meant that under appropriate conditions an RNApolymerase may bind to the promoter of the regulatory region and proceedto transcribe the nucleotide sequence such that the reporter gene istranscribed. In preferred embodiments, a reporter gene consists of theMarA family protein responsive promoter linked in frame to the reportergene. In certain embodiments, however, it may be desirable to includeother sequences, e.g, transcriptional regulatory sequences, in thereporter gene construct. For example, modulation of the activity of thepromoter may be effected by altering the RNA polymerase binding to thepromoter region, or, alternatively, by interfering with initiation oftranscription or elongation of the mRNA. Thus, sequences which areherein collectively referred to as transcriptional regulatory elementsor sequences may also be included in the reporter gene construct. Inaddition, the construct may include sequences of nucleotides that altertranslation of the resulting mRNA, thereby altering the amount ofreporter gene product.

[0053] Examples of reporter genes include, but are not limited to CAT(chloramphenicol acetyl transferase) (Alton and Vapnek (1979), Nature282: 864-869) luciferase, and other enzyme detection systems, such asbeta-galactosidase; firefly luciferase (deWet et al. (1987), Mol. Cell.Biol. 7:725-737); bacterial luciferase (Engebrecht and Silverman (1984),PNAS 1: 4154-4158; Baldwin et al. (1984), Biochemistry 23: 3663-3667);PhoA, alkaline phosphatase (Toh et al. (1989) Eur. J. Biochem. 182:231-238, Hall et al. (1983) J. Mol. Appl. Gen. 2: 101), human placentalsecreted alkaline phosphatase (Cullen and Malim (1992) Methods inEnzymol. 216:362-368) and green fluorescent protein (U.S. Pat. No.5,491,084; WO96/23898).

[0054] As used herein the term “compound” includes any reagent or testagent which is employed in the assays of the invention and assayed forits utility as an antiinfective compound based on its ability toinfluence the activity of a MarA family helix-turn-helix domain, e.g.,by binding to that domain. More than one compound, e.g., a plurality ofcompounds, can be tested at the same time for their ability to modulatethe activity of a MarA family HTH domain activity in a screening assay.

[0055] Compounds that can be tested in the subject assays includeantibiotic and non-antibiotic compounds. In one embodiment, compoundsinclude candidate detergent or disinfectant compounds. Exemplarycompounds which can be screened for activity include, but are notlimited to, peptides, non-peptidic compounds, nucleic acids,carbohydrates, small organic molecules (e.g., polyketides), and naturalproduct extract libraries. The term “non-peptidic compound” is intendedto encompass compounds that are comprised, at least in part, ofmolecular structures different from naturally-occurring L-amino acidresidues linked by natural peptide bonds. However, “non-peptidiccompounds” are intended to include compounds composed, in whole or inpart, of peptidomimetic structures, such as D-amino acids,non-naturally-occurring L-amino acids, modified peptide backbones andthe like, as well as compounds that are composed, in whole or in part,of molecular structures unrelated to naturally-occurring L-amino acidresidues linked by natural peptide bonds. “Non-peptidic compounds” alsoare intended to include natural products.

[0056] As used herein, the term “genetic loci” includes anoligonucleotide sequence encoding a peptide or a transcriptionalregulatory element (e.g., a promoter, operator, or other regulatoryelement). A locus may consist of a start codon, a stop codon, and atleast one codon encoding an amino acid residue. Typically, a locus istranscribed to produce an mRNA transcript and that transcript istranslated to produce a polypeptide.

[0057] II. MarA Family Protein Helix-Turn-Helix Domains

[0058] Helix-turn-helix domains are known in the art and have beenimplicated in DNA binding (Ann Rev. of Biochem. 1984. 53:293). Anexample of the consensus sequence for a helix-turn domain can be foundin Brunelle and Schleif (1989. J. Mol. Biol. 209:607). The domain hasbeen illustrated by the sequence XXXPhoAlaXXPhoGlyPhoXXXXPhoXXPhoXX (SEQID NO:216), where X is any amino acid and Pho is a hydrophobic aminoacid.

[0059] The crystal structure of MarA has been determined and the first(most amino terminal) HTH domain of MarA has been identified ascomprising from about amino acid 31 to about amino acid 52 and thesecond HTH domain of MarA has been identified as comprising from aboutamino acid 79 to about amino acid 102 (Rhee et al. 1998. Proc. Natl.Acad. Sci. USA. 95:10413).

[0060] Locations of the helix-turn-helix domains in other MarA familymembers can easily be found by one of skill in the art. For exampleusing the MarA protein sequence and an alignment program, e.g., theProDom program, a portion of the MarA amino acid sequence e.g.,comprising one or both HTH domains of MarA (such as from about aminoacid 30 to about amino acid 107 of MarA as was done to generate FIG. 2)to produce an alignment. Exemplary alignments are shown in FIGS. 2 and3. Using such an alignment, the amino acid sequences corresponding tothe HTH domains of MarA can be identified in other MarA family memberproteins. An exemplary consensus sequence for the first helix-turn-helixdomain of a MarA family protein can be illustrated as XXXXSXXXLXXXFX(SEQ ID NO:3), where X is any amino acid. An exemplary consensussequence for the second helix-turn-helix domain of a MarA family proteinis illustrated as XXIXXIAXXXGFXSXXXFXXX[F/Y] (SEQ ID NO:4), where X isany amino acid. Preferably, a MarA family protein first helix-turn-helixdomain comprises the consensus sequenceE/D-X-V/L-A-D/E-X-A/S-G-X—S—X3-L-Q-X2-F-K/R/E-X2-T/I (SEQ ID NO:5).Preferably, a MarA family protein second helix-turn-helix domaincomprises the consensus sequence I-X-D-I-A-X3-G-F-X-S-X2-F-X3-F-X4. (SEQID NO:6)

[0061] Preferably, a MarA family member HTH domain is a MarA HTH domain.The first and second helix-turn-helix domains of MarA are, respectively,EKVSERSGYS KWHLQRMFKKET (SEQ ID NO:208) and ILYLAE RYGFESQQTLTRTFKNYF(SEQ ID NO:209). Other exemplary MarA family helix-turn-helix domainsinclude: about amino acid 210 to about amino acid 229 and about aminoacid 259 to about amino acid 278 of MelR; about amino acid 196 to aboutamino acid 215 and about amino acid 245 to about amino acid 264 of AraC;and about amino acid 230 to about amino acid 249 (or 233-253) and aboutamino acid 281 to about amino acid 301 (or 282-302) of XylS (see e.g.,Brunelle et al. 1989. J. Mol. Biol. 209:607; Niland et al. 1996. J. Mol.Biol. 264:667; Gallegos et al. 1997. Microbiology and Molecular BiologyReviews. 61:393).

[0062] “MarA family protein helix-turn-helix domains” are derived fromor are homologous to the helix-turn-helix domains found in the MarAfamily proteins as described supra. In preferred embodiments, a MarAfamily protein excludes one or more of XylS, AraC, and MelR. Inparticularly preferred embodiments, a MarA family protein is selectedfrom the group consisting of: MarA, RamA, AarP, Rob, SoxS, and PqrA.

[0063] Both of the helix-turn-helix domains present in MarA familyproteins are in the carboxy terminal end of the protein. Proteins orportions thereof comprising either or both of these domains can be usedin the instant methods. In certain embodiments, a polypeptide which isused in screening for compounds comprises the helix-turn-helix domainmost proximal to the carboxy terminus (HTH2) of the MarA family proteinfrom which it is derived. In other embodiments, such a polypeptidecomprises the helix-turn-helix domain most proximal to the aminoterminus (HTH1) of the MarA family protein from which it is derived. Inone embodiment, other polypeptide sequences may also be present, e.g.,sequences that might facilitate immobilizing the domain on a support,or, alternatively, might facilitate the purification of the domain.

[0064] In preferred embodiments, such a polypeptide consists essentiallyof the helix-turn-helix domain most proximal to the carboxy terminus ofthe MarA family protein from which it is derived. In other preferredembodiments, such a polypeptide consists essentially of thehelix-turn-helix domain most proximal to the amino terminus of the MarAfamily protein from which it is derived.

[0065] In preferred embodiments, such a polypeptide consists of thehelix-turn-helix domain most proximal to the carboxy terminus of theMarA family protein from which it is derived. In other preferredembodiments, such a polypeptide consists of the helix-turn-helix domainmost proximal to the amino terminus of the MarA family protein fromwhich it is derived.

[0066] MarA family protein helix-turn-helix domains can be made usingtechniques which are known in the art. The nucleic acid and amino acidsequences of MarA family proteins are available, for example, fromGenBank. Using this information, the helix-turn-helix consensus motifand mutational analysis provided herein, one of ordinary skill in theart can identify MarA family helix-turn-helix domains.

[0067] In certain embodiments of the invention it will be desirable toobtain “isolated or recombinant” nucleic acid molecules encoding MarAfamily helix-turn-helix domains or mutant forms thereof. By “isolated orrecombinant” is meant a nucleic acid molecule which has been (1)amplified in vitro by, for example, polymerase chain reaction (PCR); (2)recombinantly produced by cloning, or (3) purified, as by cleavage andgel separation; or (4) synthesized by, for example, chemical synthesis.Such a nucleic acid molecule is isolated from the sequences whichnaturally flank it in the genome and from cellular components.

[0068] The isolated or recombinant nucleic acid molecules encoding MarAfamily helix-turn-helix protein domains can then, for example, beutilized in binding assays, can be expressed in a cell, or can beexpressed on the surface of phage, as discussed further below.

[0069] In yet other embodiments of the invention, it will be desirableto obtain a substantially purified or recombinant MarA familyhelix-turn-helix polypeptide. Such polypeptides, for example, can bepurified from cells which have been engineered to express an isolated orrecombinant nucleic acid molecule which encodes a MarA familyhelix-turn-helix domain. For example, as described in more detail below,a bacterial cell can be transformed with a plasmid which encodes a MarAfamily helix-turn-helix domain. The MarA family helix-turn-helix proteincan then be purified from the bacterial cells and used, for example, inthe cell-free assays described herein.

[0070] Purification of a MarA family helix-turn-helix domain can beaccomplished using techniques known in the art. For example, columnchromatography could be used, or antibodies specific for the domain orfor a polypeptide fused to the domain can be employed, for example on acolumn or in a panning assay.

[0071] In preferred embodiments, cells used to express MarA familyhelix-turn-helix domains for purification, e.g., host cells, comprise amutation which renders any endogenous MarA family member proteinnonfunctional or causes the endogenous protein to not be expressed. Inother embodiments, mutations may also be made in MarR or related genesof the host cell, such that repressor proteins which bind to the samepromoter as a MarA family protein are not expressed by the host cell.

[0072] III. Mutant MarA Family Helix-Turn-Helix Domains

[0073] In certain embodiments of the invention, it will be desirable touse a mutant form of a MarA family protein helix-turn-helix domain,e.g., a non-naturally occurring form of a MarA family helix-turn-helixdomain which has altered activity, e.g., does not retain wild type MarAfamily protein helix-turn-helix domain activity, or which has reducedactivity or which is more active when compared to a wild-type MarAfamily protein helix-turn-helix domain.

[0074] Such mutant forms can be made using techniques which are wellknown in the art. For example, random mutagenesis can be used. Usingrandom mutagenesis one can mutagenize an entire molecule or one canproceed by cassette mutagenesis. In the former instance, the entirecoding region of a molecule is mutagenized by one of several methods(chemical, PCR, doped oligonucleotide synthesis) and that collection ofrandomly mutated molecules is subjected to selection or screeningprocedures. In the second approach, discrete regions of a protein,corresponding either to defined structural or functional determinants(e.g., the first or second alpha helix of a helix-turn-helix domain) aresubjected to saturating or semi-random mutagenesis and these mutagenizedcassettes are re-introduced into the context of the otherwise wild typeallele.

[0075] In a preferred embodiment, PCR mutagenesis is used. For example,Example 2 describes the use of Megaprimer PCR(O. H. Landt, Gene96:125-128) used to introduce an NheI restriction site into the centersof both the helix A (position 1989) and helix B (position 2016) regionsof the marA gene.

[0076] In one embodiment, such mutant helix-turn-helix domains compriseone or more mutations in the helix-turn-helix domain most proximal tothe carboxy terminus (HTH2) of the MarA family protein molecule. In apreferred embodiment, the mutation comprises an insertion into helix Aand helix B of the helix-turn-helix domain most proximal to the carboxyterminus of the MarA family protein. In one embodiment, such mutanthelix-turn-helix domains comprise one or more mutations in thehelix-turn-helix domain most proximal to the amino terminus (HTH1) ofthe MarA family protein molecule. In a preferred embodiment, themutation comprises an insertion into helix A and helix B of thehelix-turn-helix domain most proximal to the amino terminus of the MarAfamily protein. In particularly preferred embodiments, the mutation isselected from the group consisting of: an insertion at an amino acidcorresponding to about position 33 of MarA and an insertion at an aminoacid position corresponding to about position 42 of MarA.“Corresponding” amino acids can be determined, e.g, using an alignmentof the helix-turn-helix domains, such as that shown in FIG. 2.

[0077] Such mutant forms of MarA family helix-turn-helix motifs areuseful as controls to verify the specificity of antiinfective compoundsfor a MarA family helix-turn-helix domain or as controls for theidentification of genetic loci which affect resistance toantiinfectives. For example, the mutant MarA family helix-turn-helixdomains described in the appended Examples demonstrate that insertionalinactivation of MarA at either helix A or helix B in the first HTHdomain abolished the multidrug resistance phenotype in both E. coli andM. smegmatis. By the use of an assay system such as that described inExample 2, which demonstrates the ability of MarA family proteinhelix-turn-helix domains to increase antibiotic resistance and thatmutant forms of these domains do not have the same effect, one canclearly show that the response of any genetic loci identified isspecific to a MarA family helix-turn-helix domain.

[0078] IV. Expression of MarA Family Helix-Turn-Helix Domains

[0079] Nucleic acids encoding MarA family protein helix-turn-helixdomains can be expressed in cells using vectors. Almost any conventionaldelivery vector can be used. Such vectors are widely availablecommercially and it is within the knowledge and discretion of one ofordinary skill in the art to choose a vector which is appropriate foruse with a given microbial cell. The sequences encoding these domainscan be introduced into a cell on a self-replicating vector or may beintroduced into the chromosome of a microbe using homologousrecombination or by an insertion element such as a transposon.

[0080] These nucleic acids can be introduced into microbial cells usingstandard techniques, for example, by transformation using calciumchloride or electroporation. Such techniques for the introduction of DNAinto microbes are well known in the art.

[0081] V. Methods of Identifying Antimicrobial/Antiinfective CompoundsWhich Interact With MarA Family Helix-Turn-Helix Domains

[0082] In one embodiment, the invention provides for methods ofidentifying an antiinfective compound which affects the activity of aMarA family helix-turn-helix domain, by contacting a polypeptidecomprising a Mar A family helix-turn-helix domain derived from a MarAfamily protein with a compound under conditions which allow interactionof the compound with the polypeptide. The ability of the compound toreduce an activity of a MarA family protein helix-turn-helix domain isused as an indication of whether the compound is an antimicrobialcompound which interferes with the ability of a microbe to grow or anantiinfective compound which interferes with the ability of a microbe tocause infection in a host.

[0083] A variety of different techniques can be used to determinewhether a compound reduces the activity of a helix-turn-helix domain.For example, the ability of a compound to decrease binding of a MarAfamily protein to DNA, e.g., to a MarA family protein responsivepromoter, or the ability of the compound to reduce MarA family proteininitiated transcription from such a promoter can be measured. Asdescribed in more detail below, either whole cell or cell free assaysystems can be employed.

[0084] A. Whole Cell Assays

[0085] In certain embodiments of the invention, the step of determiningwhether a compound affects the activity of a MarA familyhelix-turn-helix domain comprises measuring the ability of the compoundto reduce the ability of a MarA family helix-turn-helix domain toactivate transcription from a MarA responsive promoter. In such anassay, since the MarA family member helix-turn-helix domain wouldnormally bind to the MarA responsive promoter to induce transcription, acompound would be identified based on its ability to reduce this controllevel of transcription as compared to a cell which had been transfectedwith the MarA family helix-turn-helix domain but which had not beentreated with the compound.

[0086] In preferred embodiments, to provide a convenient readout of thetranscription from a MarA family protein responsive promoter, such apromoter is linked to a reporter gene. For example, a bacterial cell,e.g., an E. coli cell, can be transfected with plasmids comprising apm-lacZ reporter gene construct and a plasmid comprising XylS. XylSactivates transcription from the pm promoter under control conditionsleading to transcription and the production of reporter gene product.The ability of a compound to interfere with this interaction isindicated by a reduction in this control level of transcription and,thus, a reduction in the amount of reporter gene product. The amount ofreporter gene product can be measured grossly in intact cells, e.g., bylooking at color changes in cells, for example, by using the lacZreporter gene and plating the cells on media supplemented with X-Gal(5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside or, for example, bylysing cells and measuring the amount of product produced, e.g., byabsorbance or enzyme activity.

[0087] In yet other embodiments, the step of detecting the ability of acompound to induce a change in transcription comprises measuring theamount of RNA produced by the cell. In such embodiments, the cells mayor may not comprise a reporter gene construct. For example, the RNA canbe isolated from cells which express a MarA family helix-turn-helixdomain which have been incubated in the presence and absence ofcompound. Northern Blots using probes specific for the sequences to bedetected can then be performed using techniques known in the art.Sequences which can be detected include any sequences which are linkedto a MarA family responsive promoter, including, for example, bothendogenous sequences and reporter gene sequences. Exemplary endogenoussequences which can be detected include: acrAB; micF; mlr 1,2,3; slp;nfo; inaA; fpr; sodA; soi-17,19; zwf; fumC; or rpsF; araBAD, araE,araFGH and araC, which are activated by AraC; pm, which is activated byXylS; melAB which is activated by MelR; and oriC which is activated byRob., as well as sequences from genetic loci that are identified usingthe assays described infra.

[0088] In yet other embodiments, the ability of a compound to induce achange in transcription from a MarA responsive promoter can beaccomplished by measuring the amount of a protein produced by the cell.Proteins which can be detected include any proteins which are producedupon the activation of a MarA family responsive promoter, including, forexample, both endogenous sequences and reporter gene sequences.Exemplary endogenous proteins which can be detected include: AcrAB; Mlr1,2,3; Slp; Nfo; InaA; Fpr; SodA; Soi-17,19; Zwf; FumC; or RpsFpromoters (Alekshun and Levy. 1997. Antimicrobial Agents and Chemother.41:2067). Others are known in the art and include: AraBAD, AraE, AraFGHand AraC, which are activated by AraC; pm, which is activated by XylS;MelAB which is activated by MelR; and oriC which is bound by Rob, aswell as proteins translated from genetic loci that are identified usingthe assays described infra. In one embodiment, a the amount of proteinmade by a cell can be detected using an antibody against that protein.In other embodiments, the activity of such a protein can be measured.

[0089] B. Cell-Free Assays

[0090] In other embodiments, the ability of a compound to affect theactivity of a MarA family helix-turn-helix domain is accomplished usingisolated MarA family helix-turn-helix polypeptides in a cell-freesystem. In such an assay the step of measuring the ability of a compoundto affect the activity of the MarA family helix-turn-helix polypeptideis accomplished by measuring the effect of the compound on the abilityof helix-turn-helix domain to bind to DNA.

[0091] For example, the ability of a helix-turn-helix domain to bind toDNA can be measured by end labeling a nucleic acid molecule whichencodes for a MarA family responsive promoter with ³²P using techniqueswhich are known in the art (see e.g., Martin and Rosner. 1995. Proc.Natl. Acad. Sci. USA 92:5456). The helix-turn-helix domain can then beincubated with the compound to be tested to form a complex. The complexcan then be incubated with the labeled MarA family protein responsivepromoter. The sample can then be electrophoresed to look for changes inthe mobility of the sample as compared to the mobility of thehelix-turn-helix domain-promoter complex in the absence of the compound(Martin and Rosner, supra).

[0092] In yet another method of detecting the ability of a compound tobind a MarA family helix-turn-helix domain, the helix-turn-helix domainpolypeptide sequence can be expressed by a bacteriophage. In thisembodiment the phages which display the helix-turn-helix domain wouldthen be contacted with a compound so that the helix-turn-helix domaincan interact with and potentially form a complex with the compound.Phage which have formed complexes with compounds can then be separatedfrom those which have not. The complex of the helix-turn-helix domainand compound can then be contacted with an agent that dissociates thebacteriophage from the compound. Any compounds that bound to thehelix-turn-helix domain can then be isolated and identified.

[0093] In a variation of this method that allows for screening ofcompounds which are polypeptides and which bind to helix-turn-helixdomains, a library of bacteriophage which display on their surface aplurality of polypeptide sequences can be tested for their ability tobind a MarA family helix-turn-helix domain to obtain those polypeptideshaving affinity for the helix-turn-helix domain. The complexes of boundbacteriophage and helix-turn-helix domain can be separated, and thentreated with an agent that dissociates the bound bacteriophage from thecomplexes and the sequence of the nucleic acid encoding the displayedpolypeptide can be obtained.

[0094] VII. Microbes Suitable for Testing

[0095] Numerous different microbes are suitable for testing in theinstant assays. As such, they may be used as intact cells or as sourcesof DNA as described herein.

[0096] In preferred embodiments, microbes for use in the claimed methodsare bacteria, either Gram negative or Gram positive bacteria. Morespecifically, any bacteria that are shown to become resistant toantibiotics, e.g., to display a Mar phenotype are appropriate for use inthe claimed methods.

[0097] In preferred embodiments, microbes suitable for testing arebacteria from the family Enterobacteriaceae. In more preferredembodiments, the antiinfective is effective against a bacteria of agenus selected from the group consisting of: Escherichia, Proteus,Salmonella, Klebsiella, Providencia, Enterobacter, Burkholderia,Pseudomonas, Aeromonas, Haemophilus, Yersinia, Neisseria, andMycobacteria.

[0098] In yet other embodiments, the microbes to be tested are Grampositive bacteria and are from a genus selected from the groupconsisting of: Lactobacillus, Azorhizobium, Streptomyces, Pediococcus,Photobacterium, Bacillus, Enterococcus, Staphylococcus, Clostridium, andStreptococcus.

[0099] In other embodiments, the microbes to be tested are fungi. In apreferred embodiment the fungus is from the genus Mucor or Candida,e.g., Mucor racmeosus or Candida albicans.

[0100] In yet other embodiments, the microbes to be tested are protozoa.In a preferred embodiment the microbe is a malaria or cryptosporidiumparasite.

[0101] VIII. Test Compounds

[0102] Compounds for testing in the instant methods can be derived froma variety of different sources and can be known or can be novel. In oneembodiment, libraries of compounds are tested in the instant methods toidentify MarA family protein blocking agents. In another embodiment,known compounds are tested in the instant methods to identify MarAfamily protein blocking agents. In a preferred embodiment, compoundsamong the list of compounds generally regarded as safe (GRAS) by theEnvironmental Protection Agency are tested in the instant methods.

[0103] A recent trend in medicinal chemistry includes the production ofmixtures of compounds, referred to as libraries. While the use oflibraries of peptides is well established in the art, new techniqueshave been developed which have allowed the production of mixtures ofother compounds, such as benzodiazepines (Bunin et al. 1992. J. Am.Chem. Soc. 114:10987; DeWitt et al. 1993. Proc. Natl. Acad. Sci. USA90:6909) peptoids (Zuckernann. 1994. J. Med. Chem. 37:2678)oligocarbamates (Cho et al. 1993. Science. 261:1303), and hydantoins(DeWitt et al. supra). Rebek et al. have described an approach for thesynthesis of molecular libraries of small organic molecules with adiversity of 104-105 (Carell et al. 1994. Angew. Chem. Int. Ed. Engl.33:2059; Carell et al. Angew. Chem. Int. Ed. Engl. 1994. 33:2061).

[0104] The compounds of the present invention can be obtained using anyof the numerous approaches in combinatorial library methods known in theart, including: biological libraries; spatially addressable parallelsolid phase or solution phase libraries, synthetic library methodsrequiring deconvolution, the ‘one-bead one-compound’ library method, andsynthetic library methods using affinity chromatography selection. Thebiological library approach is limited to peptide libraries, while theother four approaches are applicable to peptide, non-peptide oligomer orsmall molecule libraries of compounds (Lam, K. S. Anticancer Drug Des.1997. 12:145).

[0105] Exemplary compounds which can be screened for activity include,but are not limited to, peptides, nucleic acids, carbohydrates, smallorganic molecules, and natural product extract libraries. In oneembodiment, the test compound is a peptide or peptidomimetic. Inanother, preferred embodiment, the compounds are small, organicnon-peptidic compounds.

[0106] Other exemplary methods for the synthesis of molecular librariescan be found in the art, for example in: Erb et al. 1994. Proc. Natl.Acad. Sci. USA 91:11422; Horwell et al. 1996 Immunopharmacology 33:68;and in Gallop et al. 1994. J. Med. Chem. 37:1233. In addition, librariessuch as those described in the commonly owned applications U.S. Ser. No.08/864,241, U.S. Ser. No. 08/864,240 and U.S. Ser. No. 08/835,623 can beused to provide compounds for testing in the present invention. Thecontents of each of these applications is expressly incorporated hereinby this reference.

[0107] Libraries of compounds may be presented in solution (e.g.,Houghten (1992) Biotechniques 13:412-421), or on beads (Lam (1991)Nature 354:82-84), chips (Fodor (1993) Nature 364:555-556), bacteria(Ladner U.S. Pat. No. 5,223,409), spores (Ladner U.S. Pat. No. '409),plasmids (Cull et al. (1992) Proc Natl Acad Sci USA 89:1865-1869) or onphage (Scott and Smith (1990) Science 249:386-390); (Devlin (1990)Science 249:404-406); (Cwirla et al. (1990) Proc. Natl. Acad. Sci.87:6378-6382); (Felici (1991) J. Mol. Biol. 222:301-310); (Ladnersupra.). Other types of peptide libraries may also be expressed, see,for example, U.S. Pat. Nos. 5,270,181 and 5,292,646). In still anotherembodiment, combinatorial polypeptides can be produced from a cDNAlibrary.

[0108] In other embodiments, the compounds can be nucleic acidmolecules. In preferred embodiments, nucleic acid molecules for testingare small oligonucleotides. Such oligonucleotides can be randomlygenerated libraries of oligonucleotides or can be specifically designedto reduce the activity of a MarA protein family member helix-turn-helixdomain. For example, in one embodiment, these oligonucleotides are senseor antisense oligonucleotides. In preferred embodiments,oligonucleotides for testing are sense to the binding site of a MarAprotein family member helix-turn-helix domain. Methods of designing sucholigonucleotides given the sequences of the MarA family member proteinhelix-turn-helix domains is within the skill of the art.

[0109] In preferred embodiments, controls should be included to ensurethat any compounds which are identified using the subject assays do notmerely appear to decrease the activity of a MarA family helix-turn-helixdomain because they inhibit protein synthesis. For example, if acompound appears to inhibit the synthesis of a protein being translatedfrom RNA which is transcribed upon activation of a MarA familyresponsive promoter, it may be desirable to show that the synthesis of acontrol, e.g., a protein which is being translated from RNA which is nottranscribed upon activation of a MarA family responsive promoter, is notaffected by the addition of the same compound. For example, the amountof the MarA family helix-turn-helix polypeptide being made or the amountof an endogenous protein could be tested. In another embodiment themicrobe could be transformed with another plasmid comprising a promoterwhich is not a MarA family responsive promoter and a protein operablylinked to that promoter. The expression of this protein could be used tonormalize the amount of protein produced in the presence and absence ofcompound.

[0110] X. Methods of Identifying Genetic Loci in an Microbe Which AffectResistance

[0111] A variety of different techniques can be used to identify newgenetic loci which are involved in mediating antibiotic resistance. Forexample, either whole cell or cell free assay systems can be employedutilizing at least one MarA family helix-turn-helix domain.

[0112] A. Cell-Based Assays

[0113] In one embodiment the invention provides cell based method ofidentifying genetic loci in an microbe which affect resistance toantibiotics. In such an assay a nucleotide sequence encoding ahelix-turn-helix motif of a MarA family protein is introduced into amicrobe and the microbe is tested for changes in its antibioticresistance profile, for example, by monitoring changes in growth inmedia containing antibiotics or by detecting a reduction in the zone ofinhibition around an antibiotic disc. The ability of a MarA familyprotein helix-turn-helix domain to decrease antibiotic sensitivity is anindication that the microbe comprises a MarA family protein responsiveendogenous genetic loci which are involved in mediating antibioticresistance.

[0114] In another embodiment, the above method can further involveassaying for changes in transcription of the genetic loci identified inthe microbe. For example, a test microbe can be transformed with avector bearing a MarA family helix-turn-helix domain. Suitable controlmicrobes include those which lack any such heterologous DNA or aretransformed with a vector bearing a mutant form of a MarA familyhelix-turn-helix domain. The total RNA from the test and controlmicrobes can be isolated. This can be done, for example, by making acDNA library from both of the strains. The cDNAs from the test andcontrol strains can then be incubated together under conditions whichare favorable to hybridization. cDNAs which do not hybridize and remainsingle stranded may be involved in mediating antibiotic resistance andcan be isolated and sequenced using standard techniques.

[0115] In another example of a method by which the total mRNA from cellsbearing a MarA family helix-turn-helix domain can be compared to cellswith lack such a domain or bear a mutant form of such a domain, a cDNAlibrary can be made from the total RNA of these cells. This cDNA librarycan be used to generate labeled probes which can be used in a standardNorthern blot screen. Any cDNA probes that hybridize to the mRNA of thecells comprising a MarA family helix-turn-helix domain, but not to themRNA from control cells will be involved in mediating antibioticresistance. Once these cDNA probes which specifically hybridize to cellscomprising a MarA family helix-turn-helix domain are identified, theseprobes can be used to identify genes involved in mediating antibioticresistance using standard techniques.

[0116] A. Cell-Free Assays

[0117] In other embodiments of the invention, MarA family proteinresponsive genetic loci involved in mediating antibiotic resistance areidentified using cell-free assays. In one embodiment, a cell-free methodof identifying such genetic loci involves contacting a nucleic acidmolecule of the microbe with a MarA family protein helix-turn-helixdomain and allowing complexes to form. The helix-turn-helixdomain-nucleic acid molecule complexes are separated from theuncomplexed helix-turn-helix domains and the sequence of those nucleicacid molecules which can bind to a MarA family protein helix-turn-helixdomain can then be sequenced to identify loci involved in mediatingantibiotic resistance.

[0118] For example, substantially purified MarA family proteinhelix-turn-helix domain polypeptide is mixed with the fragmented genomicDNA of an microbe under conditions which permit the polypeptide to bindto appropriate DNA sequences. DNA fragments to which thehelix-turn-helix domain has bound can be isolated using a column,filters, polyacrylamide gels, or any other methods well known to thoseof ordinary skill in the art. The DNA which has bound to thehelix-turn-helix domain can then be released from the domain and clonedinto vectors or used as probes to locate and isolate the genes to whichthey correspond. Any such genes can then be sequenced.

[0119] In another aspect the invention also provides for kits foridentifying genetic loci in an microbe which affect resistance tocompounds. Such kits comprise a nucleotide sequence encoding a MarAfamily protein helix-turn-helix domain and/or substantially purifiedMarA family protein helix-turn-helix domains and nucleotide sequenceencoding a mutant form of a MarA family protein helix-turn-helix domainand/or substantially purified mutant forms of a MarA family proteinhelix-turn-helix domain. By providing both functional MarA familyprotein helix-turn-helix domains and mutant forms of such domains, thesubject kits provide both the test and control reagents which facilitateboth optimal performance of the claimed methods and optimalinterpretation of results.

[0120] XI. Formulations Comprising Compounds Identified in the InstantAssays

[0121] The invention provides pharmaceutically acceptable compositionswhich include a therapeutically-effective amount or dose of a compoundidentified in any of the instant assays and one or more pharmaceuticallyacceptable carriers (additives) and/or diluents. A composition can alsoinclude a second antimicrobial agent, e.g., an antibiotic.

[0122] As described in detail below, the pharmaceutical compositions canbe formulated for administration in solid or liquid form, includingthose adapted for the following: (1) oral administration, for example,aqueous or non-aqueous solutions or suspensions, tablets, boluses,powders, granules, pastes; (2) parenteral administration, for example,by subcutaneous, intramuscular or intravenous injection as, for example,a sterile solution or suspension; (3) topical application, for example,as a cream, ointment or spray applied to the skin; (4) intravaginally orintrarectally, for example, as a pessary, cream, foam, or suppository;or (5) aerosol, for example, as an aqueous aerosol, liposomalpreparation or solid particles containing the compound.

[0123] The phrase “pharmaceutically-acceptable carrier” as used hereinmeans a pharmaceutically-acceptable material, composition or vehicle,such as a liquid or solid filler, diluent, excipient, solvent orencapsulating material, involved in carrying or transporting theantiinfective agents or compounds of the invention from one organ, orportion of the body, to another organ, or portion of the body withoutaffecting its biological effect. Each carrier should be “acceptable” inthe sense of being compatible with the other ingredients of thecomposition and not injurious to the subject. Some examples of materialswhich can serve as pharmaceutically-acceptable carriers include: (1)sugars, such as lactose, glucose and sucrose; (2) starches, such as cornstarch and potato starch; (3) cellulose, and its derivatives, such assodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate;(4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8)excipients, such as cocoa butter and suppository waxes; (9) oils, suchas peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil,corn oil and soybean oil; (10) glycols, such as propylene glycol; (11)polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol;(12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14)buffering agents, such as magnesium hydroxide and aluminum hydroxide;(15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18)Ringer's solution; (19) ethyl alcohol; (20) phosphate buffer solutions;and (21) other non-toxic compatible substances employed inpharmaceutical compositions. Proper fluidity can be maintained, forexample, by the use of coating materials, such as lecithin, by themaintenance of the required particle size in the case of dispersions,and by the use of surfactants.

[0124] These compositions may also contain adjuvants such aspreservatives, wetting agents, emulsifying agents and dispersing agents.Prevention of the action of microbes may be ensured by the inclusion ofvarious antibacterial and antifungal agents, for example, paraben,chlorobutanol, phenol sorbic acid, and the like. It may also bedesirable to include isotonic agents, such as sugars, sodium chloride,and the like into the compositions. In addition, prolonged absorption ofthe injectable pharmaceutical form may be brought about by the inclusionof agents which delay absorption such as aluminum monostearate andgelatin.

[0125] In some cases, in order to prolong the effect of a drug, it isdesirable to slow the absorption of the drug from subcutaneous orintramuscular injection. This may be accomplished by the use of a liquidsuspension of crystalline or amorphous material having poor watersolubility. The rate of absorption of the drug then depends upon itsrate of dissolution which, in turn, may depend upon crystal size andcrystalline form. Alternatively, delayed absorption of aparenterally-administered drug form is accomplished by dissolving orsuspending the drug in an oil vehicle.

[0126] Pharmaceutical compositions of the present invention may beadministered to epithelial surfaces of the body orally, parenterally,topically, rectally, nasally, intravaginally, intracisternally. They areof course given by forms suitable for each administration route. Forexample, they are administered in tablets or capsule form, by injection,inhalation, eye lotion, ointment, etc., administration by injection,infusion or inhalation; topical by lotion or ointment; and rectal orvaginal suppositories.

[0127] The phrases “parenteral administration” and “administeredparenterally” as used herein mean modes of administration other thanenteral and topical administration, usually by injection, and includes,without limitation, intravenous, intramuscular, intraarterial,intrathecal, intracapsular, intraorbital, intracardiac, intradermal,intraperitoneal, transtracheal, subcutaneous, subcuticular,intraarticular, subcapsular, subarachnoid, intraspinal and intrasternalinjection and infusion.

[0128] The phrases “systemic administration,” “administeredsystemically,” “peripheral administration” and “administeredperipherally” as used herein mean the administration of a sucroseoctasulfate and/or an antibacterial, drug or other material other thandirectly into the central nervous system, such that it enters thesubject's system and, thus, is subject to metabolism and other likeprocesses, for example, subcutaneous administration.

[0129] In some methods, the compositions of the invention can betopically administered to any epithelial surface. An “epithelialsurface” according to this invention is defined as an area of tissuethat covers external surfaces of a body, or which lines hollowstructures including, but not limited to, cutaneous and mucosalsurfaces. Such epithelial surfaces include oral, pharyngeal, esophageal,pulmonary, ocular, aural, nasal, buccal, lingual, vaginal, cervical,genitourinary, alimentary, and anorectal surfaces.

[0130] Compositions can be formulated in a variety of conventional formsemployed for topical administration. These include, for example,semi-solid and liquid dosage forms, such as liquid solutions orsuspensions, suppositories, douches, enemas, gels, creams, emulsions,lotions, slurries, powders, sprays, lipsticks, foams, pastes,toothpastes, ointments, salves, balms, douches, drops, troches, chewinggums, lozenges, mouthwashes, rinses.

[0131] Conventionally used carriers for topical applications includepectin, gelatin and derivatives thereof, polylactic acid or polyglycolicacid polymers or copolymers thereof, cellulose derivatives such asmethyl cellulose, carboxymethyl cellulose, or oxidized cellulose, guargum, acacia gum, karaya gum, tragacanth gum, bentonite, agar, carbomer,bladderwrack, ceratonia, dextran and derivatives thereof, ghatti gum,hectorite, ispaghula husk, polyvinypyrrolidone, silica and derivativesthereof, xanthan gum, kaolin, talc, starch and derivatives thereof,paraffin, water, vegetable and animal oils, polyethylene, polyethyleneoxide, polyethylene glycol, polypropylene glycol, glycerol, ethanol,propanol, propylene glycol (glycols, alcohols), fixed oils, sodium,potassium, aluminum, magnesium or calcium salts (such as chloride,carbonate, bicarbonate, citrate, gluconate, lactate, acetate, gluceptateor tartrate).

[0132] Such compositions can be particularly useful, for example, fortreatment or prevention of an unwanted cell, e.g., vaginal Neisseriagonorrhoeae, or infections of the oral cavity, including cold sores,infections of eye, the skin, or the lower intestinal tract. Standardcomposition strategies for topical agents can be applied to theantiinfective compounds or a pharmaceutically acceptable salt thereof inorder to enhance the persistence and residence time of the drug, and toimprove the prophylactic efficacy achieved.

[0133] For topical application to be used in the lower intestinal tractor vaginally, a rectal suppository, a suitable enema, a gel, anointment, a solution, a suspension or an insert can be used. Topicaltransdermal patches may also be used. Transdermal patches have the addedadvantage of providing controlled delivery of the compositions of theinvention to the body. Such dosage forms can be made by dissolving ordispersing the agent in the proper medium.

[0134] Compositions of the invention can be administered in the form ofsuppositories for rectal or vaginal administration. These can beprepared by mixing the agent with a suitable non-irritating carrierwhich is solid at room temperature but liquid at rectal temperature andtherefore will melt in the rectum or vagina to release the drug. Suchmaterials include cocoa butter, beeswax, polyethylene glycols, asuppository wax or a salicylate, and which is solid at room temperature,but liquid at body temperature and, therefore, will melt in the rectumor vaginal cavity and release the active agent.

[0135] Compositions which are suitable for vaginal administration alsoinclude pessaries, tampons, creams, gels, pastes, foams, films, or spraycompositions containing such carriers as are known in the art to beappropriate. The carrier employed in the sucroseoctasulfate/contraceptive agent should be compatible with vaginaladministration and/or coating of contraceptive devices. Combinations canbe in solid, semi-solid and liquid dosage forms, such as diaphragm,jelly, douches, foams, films, ointments, creams, balms, gels, salves,pastes, slurries, vaginal suppositories, sexual lubricants, and coatingsfor devices, such as condoms, contraceptive sponges, cervical caps anddiaphragms.

[0136] For ophthalmic applications, the pharmaceutical compositions canbe formulated as micronized suspensions in isotonic, pH adjusted sterilesaline, or, preferably, as solutions in isotonic, pH adjusted sterilesaline, either with or without a preservative such as benzylalkoniumchloride. Alternatively, for ophthalmic uses, the compositions can beformulated in an ointment such as petrolatum. Exemplary ophthalmiccompositions include eye ointments, powders, solutions and the like.

[0137] Powders and sprays can contain, in addition to sucroseoctasulfate and/or antibiotic or contraceptive agent(s), carriers suchas lactose, talc, aluminum hydroxide, calcium silicates and polyamidepowder, or mixtures of these substances. Sprays can additionally containcustomary propellants, such as chlorofluorohydrocarbons and volatileunsubstituted hydrocarbons, such as butane and propane.

[0138] Ordinarily, an aqueous aerosol is made by formulating an aqueoussolution or suspension of the agent together with conventionalpharmaceutically acceptable carriers and stabilizers. The carriers andstabilizers vary with the requirements of the particular compound, buttypically include nonionic surfactants (Tweens, Pluronics, orpolyethylene glycol), proteins like serum albumin, sorbitan esters,oleic acid, lecithin, amino acids such as glycine, buffers, salts,sugars or sugar alcohols. Aerosols generally are prepared from isotonicsolutions.

[0139] Compositions of the invention can also be orally administered inany orally-acceptable dosage form including, but not limited to,capsules, cachets, pills, tablets, lozenges (using a flavored basis,usually sucrose and acacia or tragacanth), powders, granules, or as asolution or a suspension in an aqueous or non-aqueous liquid, or as anoil-in-water or water-in-oil liquid emulsion, or as an elixir or syrup,or as pastilles (using an inert base, such as gelatin and glycerin, orsucrose and acacia) and/or as mouth washes and the like, each containinga predetermined amount of sucrose octasulfate and/or antibiotic orcontraceptive agent(s) as an active ingredient. A compound may also beadministered as a bolus, electuary or paste. In the case of tablets fororal use, carriers which are commonly used include lactose and cornstarch. Lubricating agents, such as magnesium stearate, are alsotypically added. For oral administration in a capsule form, usefuldiluents include lactose and dried corn starch. When aqueous suspensionsare required for oral use, the active ingredient is combined withemulsifying and suspending agents. If desired, certain sweetening,flavoring or coloring agents may also be added.

[0140] Tablets, and other solid dosage forms, such as dragees, capsules,pills and granules, may be scored or prepared with coatings and shells,such as enteric coatings and other coatings well known in thepharmaceutical-formulating art. They may also be formulated so as toprovide slow or controlled release of the active ingredient thereinusing, for example, hydroxypropylmethyl cellulose in varying proportionsto provide the desired release profile, other polymer matrices,liposomes and/or microspheres. They may be sterilized by, for example,filtration through a bacteria-retaining filter, or by incorporatingsterilizing agents in the form of sterile solid compositions which canbe dissolved in sterile water, or some other sterile injectable mediumimmediately before use. These compositions may also optionally containopacifying agents and may be of a composition that they release theactive ingredient(s) only, or preferentially, in a certain portion ofthe gastrointestinal tract, optionally, in a delayed manner. Examples ofembedding compositions which can be used include polymeric substancesand waxes. The active ingredient can also be in micro-encapsulated form,if appropriate, with one or more of the above-described excipients.

[0141] Liquid dosage forms for oral administration includepharmaceutically acceptable emulsions, microemulsions, solutions,suspensions, syrups and elixirs. In addition to the active ingredient,the liquid dosage forms may contain inert diluents commonly used in theart, such as, for example, water or other solvents, solubilizing agentsand emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethylcarbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propyleneglycol, 1,3-butylene glycol, oils (in particular, cottonseed, groundnut,corn, germ, olive, castor and sesame oils), glycerol, tetrahydrofurylalcohol, polyethylene glycols and fatty acid esters of sorbitan, andmixtures thereof.

[0142] Besides inert diluents, the oral compositions can also includeadjuvants such as wetting agents, emulsifying and suspending agents,sweetening, flavoring, coloring, perfuming and preservative agents.

[0143] Suspensions, in addition to the antiinfective agent(s) maycontain suspending agents as, for example, ethoxylated isostearylalcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystallinecellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth,and mixtures thereof.

[0144] Sterile injectable forms of the compositions of this inventioncan be aqueous or oleaginous suspension. These suspensions may beformulated according to techniques known in the art using suitabledispersing or wetting agents and suspending agents. Wetting agents,emulsifiers and lubricants, such as sodium lauryl sulfate and magnesiumstearate, as well as coloring agents, release agents, coating agents,sweetening, flavoring and perfuming agents, preservatives andantioxidants can also be present in the compositions.

[0145] The sterile injectable preparation may also be a sterileinjectable solution or suspension in a nontoxic parenterally-acceptablediluent or solvent, for example as a solution in 1,3-butanediol. Amongthe acceptable vehicles and solvents that may be employed are water,Ringer's solution and isotonic sodium chloride solution. In addition,sterile, fixed oils are conventionally employed as a solvent orsuspending medium. For this purpose, any bland fixed oil may be employedincluding synthetic mono-or di-glycerides. Fatty acids, such as oleicacid and its glyceride derivatives are useful in the preparation ofinjectables, as are natural pharmaceutically-acceptable oils, such asolive oil or castor oil, especially in their polyoxyethylated versions.These oil solutions or suspensions may also contain a long-chain alcoholdiluent or dispersant, such as Ph. Helv or similar alcohol.

[0146] The antiinfective agent or a pharmaceutically acceptable saltthereof will represent some percentage of the total dose in other dosageforms in a material forming a combination product, including liquidsolutions or suspensions, suppositories, douches, enemas, gels, creams,emulsions, lotions slurries, soaps, shampoos, detergents, powders,sprays, lipsticks, foams, pastes, toothpastes, ointments, salves, balms,douches, drops, troches, lozenges, mouthwashes, rinses and others.Creams and gels for example, are typically limited by the physicalchemical properties of the delivery medium to concentrations less than20% (e.g., 200 mg/gm). For special uses, far less concentratedpreparations can be prepared, (e.g., lower percent formulations forpediatric applications). For example, the pharmaceutical composition ofthe invention can comprise sucrose octasulfate in an amount of0.001-99%, typically 0.01-75%, more typically 0.1-20%, especially 1-10%by weight of the total preparation. In particular, a preferredconcentration thereof in the preparation is 0.5-50%, especially 0.5-25%,such as 1-10%. It can be suitably applied 1-10 times a day, depending onthe type and severity of the condition to be treated or prevented.

[0147] Given the low toxicity of an antiinfective agent or apharmaceutically acceptable salt thereof over many decades of clinicaluse as an anti-ulcerant [W. R. Garnett, Clin. Pharm. 1:307-314 (1982);R. N. Brogden et al., Drugs 27:194-209 (1984); D. M. McCarthy, New Eng JMed., 325:1017-1025 (1991), an upper limit for the therapeuticallyeffective dose is not a critical issue.

[0148] For prophylactic applications, the pharmaceutical composition ofthe invention can be applied prior to potential infection. The timing ofapplication prior to potential infection can be optimized to maximizethe prophylactic effectiveness of the compound. The timing ofapplication will vary depending on the mode of administration, theepithelial surface to which it is applied, the surface area, doses, thestability and effectiveness of composition under the pH of theepithelial surface, the frequency of application, e.g., singleapplication or multiple applications. One skilled in the art will beable to determine the most appropriate time interval required tomaximize prophylactic effectiveness of the compound.

[0149] The practice of the present invention will employ, unlessotherwise indicated, conventional techniques of cell biology, cellculture, molecular biology, microbiology, recombinant DNA, andimmunology, which are within the skill of the art. Such techniques areexplained fully in the literature. See, for example, Genetics; MolecularCloning A Laboratory Manual, 2nd Ed., ed. by Sambrook, J. et al. (ColdSpring Harbor Laboratory Press (1989)); Short Protocols in MolecularBiology, 3rd Ed., ed. by Ausubel, F. et al. (Wiley, NY (1995)); DNACloning, Volumes I and II (D. N. Glover ed., 1985); OligonucleotideSynthesis (M. J. Gait ed. (1984)); Mullis et al. U.S. Pat. No.4,683,195; Nucleic Acid Hybridization (B. D. Hames & S. J. Higgins eds.(1984)); the treatise, Methods In Enzymology (Academic Press, Inc.,N.Y.); Immunochemical Methods In Cell And Molecular Biology (Mayer andWalker, eds., Academic Press, London (1987)); Handbook Of ExperimentalImmunology, Volumes I-IV (D. M. Weir and C. C. Blackwell, eds. (1986));and Miller, J. Experiments in Molecular Genetics (Cold Spring HarborPress, Cold Spring Harbor, N.Y. (1972)).

[0150] The contents of all references, pending patent applications andpublished patents, cited throughout this application are herebyexpressly incorporated by reference. In addition, the contents of U.S.Pat. No. 5,650,321 are also expressly incorporated by this reference.

[0151] The invention is further illustrated by the following examples,which should not be construed as further limiting.

EXAMPLES Example 1 The Identification of Genetic Loci in Mycobacreriumsmegmatis Which are Involved in Antibiotic Resistance

[0152] Multidrug resistance in Mycobacterium is presumed to occur viathe accumulation of independent chromosomal mutations which affectsusceptibility to individual drugs or a single plieotropic mutation,e.g., in mar. In Escherichia coli and other Enterobacteriaceae multidrugresistance is generally attributed to plasmids and transposons. Still,multidrug resistance can arise via derepression of the E. coli mar(multiple antibiotic resistance) operon, either by mutation or exposureto inducing compounds (S. P. Cohen et al, J. Bacteriol., 1993,175:1484-1492). In Mycobacterium, the observed relatively high frequencyof multidrug resistance and the suggested relationship of inadequatetreatment to the emergence of resistance (B. R. Bloom et al, Science,1992, 257:10544-10642) fit with the selection of E. coli Mar mutants.The possible existence of a mar-like regulatory drug resistance responsein Mycobacterium smegmatis antimicrobial susceptibility in cellsexpressing the cloned E. coli marA gene was examined.

[0153] PCR oligonucleotide primers were used to prepare a wild-type marAamplicon from E. coli AG100 (A. M. George, et al, J. Bacteriol., 1983,155:531-540) chromosomal DNA, based on the annotated sequence (S. P.Cohen, et al. J. Bacteriol., 1993, 175:1484-1492). The oligonucleotideprimers corresponded with nucleotide positions 1893 to 1910 and 2265 to2282 and contained terminal EcORI restriction enzyme sites to allowinsertion of marA in frame with the hsp60 mycobacterial heat shockpromoter resident on the E. coli-Mycobacterium shuttle plasmid pMV261(W. R. Jacobs, et al. Microbiol. Immunol., 1990, 155:153-160). The“megaprimer” PCR method (O. H. Landt, et al., Gene., 1990, 96:125-128)was used to create insertional mutants of marA in the center of eachalpha-helical region of the putative helix-turn-helix (HTH) domain ofMarA (FIG. 1). These mutant marA genes were ligated to pMV261 and pET13afor testing in M. smegmatis and E. coli, respectively. Plasmids wereintroduced into E. coli and M. smegmatis mc²155 by electroporation witha Gene Pulser transfection apparatus (Bio-Rad. Richmond, Calif.) andselected on kanamycin (10 or 25 μg/ml).

[0154] Cultures of M. smegmatis mc²¹⁵⁵ with and without plasmids weregrown at 30 or 37° C. by using 7H9 or 7H10 Middlebrook medium (Difco)enriched with Middlebrook Dubos albumic complex supplement (OADC),respectively, supplemented with 0.05% Tween 80 and with kanamycin (10μg/ml) where appropriate to maintain the Kan^(r) plasmids. Antimicrobialsusceptibilities were tested without kanamycin in 7H10-OADC antibioticgradient plates (M. S. Curial, et al. J. Bacteriol., 1982, 151:209-215)at 30 and 37° C. Tetracycline, chloramphenicol, norfloxacin, andphenazine methosulfate were purchased from Sigma Chemical Co. (St.Louis, Mo.). Isoniazid, rifampin, streptomycin sulfate, and ethambutolwere kindly provided by J. Crawford (Centers for Disease Control andPrevention, Atlanta. Ga.), and sparfloxacin was received fromRhone-Poulenc (Paris, France).

[0155]M. smegmatis mc²155 bearing pMV261:marA showed increasedresistance to multiple antimicrobial agents, including rifampin,isoniazid, ethambutol, chloramphenicol, and tetracycline, compared tothe microbe with vector alone (Table 3) when grown at 37° C. but not at30° C. Increased resistance to rifampin, however, was also noted at 30°C. Rifampin resistance also increased in the presence of vector alone atboth temperatures, although this finding was variable. When it didoccur, this was the only drug to which the vector appeared to affectparental susceptibility levels. Resistance of M. smegmatis tochloramphenicol increased two-fold and resistance to tetracyclineincreased nearly five-fold in the presence of marA. Ethambutol andisoniazid resistance increased 1.5- and 2.5-fold at 37° C. Little if anychange in susceptibility to nalidixic acid, phenazine methosulfate, orsparfloxacin occurred; some increased susceptibility was observed fornorfloxacin and streptomycin.

[0156] These changes in drug susceptibility were not seen with the marAgene cloned in the reverse orientation relative to the mycobacterialhsp60 promoter. Also, introduction of marR cloned with the same vectorby PCR methods (primer nucleotide positions 1446 to 1462 and 1864 to1879) caused no changes in susceptibility of M. smegmatis to any of thecompounds tested (Table 3). Strains selected for spontaneous loss ofplasmids by growth in the absence of kanamycin showed a return of thewild-type susceptibility phenotype. While multidrug resistance wasclearly temperature dependent, and correlated with the presence of marAbehind the heat shock promoter, it could reflect a resistancemechanism(s) per se which functions better at 37° C. than at 30° C.regardless of MarA expression. Of note, however, notemperature-dependent differences in susceptibility of wild-type cellswere observed with any of the agents tested (Table 3).

Example 2 Demonstration That the Resistance Phenotype in M. smegmatiswas a Direct Result of MarA Activity in the Cell as Demonstrated byInsertional Mutants Targeted to the Predicted Helix-Turn-Helix Domain

[0157] To obtain support for the notion that the resistance phenotypewas a direct result of MarA activity in the cell, insertional mutantstargeted to the first helix-turn-helix domain (HTH1) (M. N. Alekshun, etal. Chemother, 41:2067-2075) of the MarA protein were constructed.Megaprimer PCR(O. H. Landt, Gene 96:125-128) was used to introduce anNheI restriction site into the centers of both the helix A (position1989) and helix B (position 2016) regions of marA. A double-strandedsynthetic oligonucleotide with compatible ends was ligated to the NheIsites to produce two distinct insertional mutants interrupting each ofthe two putative alpha-helical regions (FIG. 1). These mutated geneswere cloned into pMV261, as described above, for susceptibility testingin M. smegmatis at 37° C. They were also expressed from theisopropyl-β-D-thiogalactopyranoside (IPTG)-regulated T7 promoterresident on plasmid pET13a for testing in E. coli BL21 (Studier et al.1990. Methods Enzymol. 185:60). Insertional inactivation of MarA ateither helix A or helix B of the first HTH abolished the multidrugresistance phenotype in both E. coli and M. smegmatis the (Table 3 and4).

[0158] To conform MarA expression, Northern blotting (S. K. Goda,Nucleic Acids Res. 23:3357-3358) was performed with total cellular RNAisolated by the TRIzol method (Gibco BRL, Gaithersburg, Md.) frommid-log-phase cells grown at 30 and 37° C. in Middlebrook 7H9-OADCmedium following 1 h of pretreatment with lysozyme (4 mg of Tris-EDTA,pH 8.0, per ml) at 30° C. Equal amounts of RNA separatedelectrophoretically in 20 mM guanidine isothiocyanate, were probed witha radiolabeled marA PCR product. Hybridization signals were visualizedwith a PhosphorImager (Molecular Dynamics, Sunnyvale, Calif.).

[0159] MarA expression was observed in cells carrying the wild-type E.coli marA gene but not in the host carrying vector. Northern analysiswas performed with PCR-amplified marA probe (nucleotide primers 1910 to1893 and 2265 to 2282). The intensity of the marA hybridization signalwas approximately fivefold higher in cells grown at 37 than at 30° C. Asexpected, a hybridization signal was detected in vector carrying marA inthe reverse orientation, since a double-stranded marA probe was used.

[0160] Anti-MarA antiserum was prepared with MarA purified fromBL21(DE3)pLysS cells (F. W. Studier, et al., Methods Enzymol, 1990,185:60-89) bearing marA (M. N. Alekshun, Antimicrob. Agents Chemother,1997, 41:2067-2075) cloned under the control of the T7 RNA polymeraseinitiation signals of pET13a. After induction with IPTG for 30 minutes,rifampin was added to maximize MarA synthesis. MarA was purified by acombination of the procedures of Li and Demple (Z. Li, et al, 1994,Biol. Chem., 1994, 269:18371-18377) and Langley et al. (K. E. Langley,et al., 1987, Euro. J. Biochem., 163:313-321). Anti-MarA rabbitantiserum was generated with purified MarA by Biodesign International(Kennebunk. Me.).

[0161] For Western analysis, cell lysates were prepared frommid-log-phase M smegmatis or E. coli cultures by sonication in buffer(10 mM Tris-HCl, pH 8.0; 3C7O sodium dodecyl sulfate) on ice. Prior toelectrophoresis. samples were treated by boiling for 5 min in samplebuffer (125 mM Tris-HCl, pH 6.8; 20% glycerol; 6 mM α-mercaptoethanol:0.05% bromphenol blue), and equivalent amounts of total protein wereresolved by electrophoresis in a sodium dodecyl sulfate-17.5%polyacrylamide gel electrophoresis gel. Each lane contains 15 μg ofmycobacteral protein from supernatant fractions. Proteins from E. coliAG100 and AG102 were used as negative and positive controls. Proteinswere transferred to Immobilon-P membranes (Amersham) and analyzed byusing rabbit anti-MarA antiserum and chemiluminescent detection (with akit from New England Biolabs, Beverly Mass.).

[0162] A protein band migrating to the same place as purified MarA andhaving the expected molecular mass (14.3 kDa) was detected inMarA-containing cells grown at both temperatures (FIG. 2B); however,considerably more MarA was produced in cells incubated at 37° C. Sincesmall amounts of MarA were detected at 30° C. (FIG. 2B), the variableresistance to rifampin and the increased susceptibility phenotypes at30° C. may have been produced by relatively low cytoplasmic levels ofMarA protein. By the same Western analysis. MarA was easily detected inE. coli and M smegmatis lysates containing the mutant marA genes.

[0163] The mechanism of MarA-mediated multidrug resistance inMycobacterium is unknown. The lack of a resistance phenotype mediated bythe two different expressed mutant MarA proteins suggests that themultidrug resistance observed resulted from direct transcriptionalactivation of cognate promoters by MarA in M. smegmatis. Alternatively,MarA may have acted indirectly through induction of, or interactionwith, endogenous proteins that mediate the mycobacterial Mar phenotype.In both instances. the multidrug resistance phenotype would haveresulted from a mar-like regulatory system operating on other genes inthis microbe. The effect, as with MarA in E. coli, may be linked toactivation of a yet-to-be-discovered multidrug efflux system. Thepresence of efflux-like proteins (J. L. Doran, et al., 1997, Clin.Diagn. Lab. Immunol., 4:23-32 and J. H. Lui, et al., J. Bacteriol.,1996, 178:3791-3795), along with the earlier report of the existence ofmycobacterial porin proteins (S. D. Mukhopadhyay, et al., J. Bacteriol.,1997, 179:6205-6207 and J. V. Trias, et al., Science, 1992,258:1479-1481), indicate that, like in E. coli, effector proteins formar-like multidrug resistance are present in Mycobacterium. The recentlycompleted Mycobacterium tuberculosis genome sequencing project (W. J.Philipp, et al., Proc. Natl. Acad. Sci., 1996, 93:3132-3137) identifiedat least two proteins similar to MarA. Determination of whether theseelements represent an endogenous mar-like system in this species awaitsfurther study.

[0164] In addition to defining a function for MarA in a heterologousgenus, our results are the first direct evidence of structurallyimportant regions of MarA. The helix-turn-helix region targeted insite-directed mutagenesis corresponds to regions in the homologousproteins AraC and XylS (M. T. Gallegos, et al., Microbiol. Mol. Biol.,Rev., 1997, 61:393-410), which are involved in DNA binding andtranscriptional activation (A. Brunelle, et al. J. Mol. Biol., 1989,209:607-622 and M. T. Gallegos, et al., J. Bacteriol, 1996,178:6427-6434), Although the insertional mutations reported here involvesignificant changes to the wild-type protein, they point to thepredicted helix-turn-helix domain as critical for protein function

Example 3 Development of a Reporter Gene Screening Assay for IdentifyingCompounds That Reduce the Activity of a MarA Family ProteinHelix-Turn-Helix Domain

[0165] The MarA family helix-turn-helix domain described in the previousexamples is expressed in E. coli along with an inaA1::phoA reporterconstruct, made as previously described for inaA1:lacZ (Martin et al.1995. J. Bacteriol. 177:4176), or a micf:lacZ reporter gene construct.The cells containing the phoA reporter construct are plated on mediasupplemented with 5-bromo-4-chloro-3-indolyl phosphate and containingkanamycin, while the cells containing the lacZ marker are grown on5-bromo-4-chloro-3-indolyl β-D-galactoside with kanamycin. Colonieswhich turn blue, indicating transcription of the reporter geneconstruct, are isolated and placed in suspension. These cells aredivided into two populations for treatment with each compound to beassayed: one test population to be treated with test compound and acontrol population to remain untreated. The appropriate population ofcells are contacted with each compound to be tested. The cells areplated onto the same selective medium as indicated above. Colonies whichturn blue indicate that the compound has no effect on the ability of theMarA helix-turn-helix domain to activate transcription of the reportergene construct, while colonies which remain white after treatment with acompound indicate that the compound reduces the activity of the MarAhelix-turn-helix domain.

Example 4 Development of a Screening Assay for Identifying CompoundsThat Increase the Antibiotic Sensitivity of an Organism Bearing a MarAFamily Protein Helix-Turn-Helix Domain

[0166]M. smegmatis mc²155 bearing pMV261::marA, which was shown above tohave increased resistance to the antimicrobial agents: rifampin,isoniazid, ethambutol, chloramphenicol, and tetracycline is used to testfor compounds which decrease this resistance. These cells are dividedinto two populations for treatment with each compound to be assayed: onetest population to be treated with test compound and a controlpopulation to remain untreated. The appropriate population of cells iscontacted with each compound to be tested. The treated and untreatedcells are plated onto plates containing medium. Antibiotic sensitivitydiscs for the antibiotics listed above are placed on the plated cellsand the plates are incubated. Compounds which reduce the zone ofantibiotic sensitvity, i.e., show a larger zone of growth inhibitionaround the antibiotic discs from that seen in the cells which are nottreated with compound are selected for identification.

Example 5 Analysis of MarA Expressed Transcripts Using DNA “Chip”(GeneChip®) Technology

[0167] To identify all MarA induced/regulated transcripts in Escherichiacoli in vivo, DNA “chip” (GeneChip®) technology is employed. DNAcomputer chips containing the entire E. coli chromosome are availablethrough Affymetrix (Santa Clara, Calif.). In brief, E. coli containing amarA expression vector is induced in order to overexpress MarA in vivo.This treatment results in the activation of MarA regulated promoters.Total cDNA is prepared from these cells and used to probe the DNA chips.As a control, cDNA prepared from E. coli containing the expressionvector lacking marA is used. This approach is also used to identify allgenes expressed following exposure to any compound that induces marexpression.

Example 6 Negative Antisense Control of MarA Regulated Loci

[0168] Negative regulation of MarA responsive transcripts is achievedusing a method similar to a previously described protocol (White et al.,Antimicrob. Agents Chemother. 41:2699). E. coli is transformed with avector encoding antisense-oligonucleotides complementary to 5′ portionsof the marA, rob, soxS, or other MarA family member protein transcriptsfollowing expression of these antisense-oligonucleotides in vivo. Thesestructures interfere with translation of the marA, rob, soxS, etc.transcripts are targeted and degraded by endogenous RNaseH, an enzymethat degrades RNA-DNA hybrids. A vector which encodesantisense-oligonucleotides targeted toward all of the MarA homologs inE. coli is designed. Transfection of this vector into E. coli diminishesor eliminates the host's MarA family member regulated adaptationalresponse to many antibiotics, disinfectants, orgainc solvents, and/orother environmental stimuli.

Example 7 Resistance Pattern of E. coli Microbes with Plasmids BearingDifferent E. coli Mar Genes

[0169] Antibiotic MICs were determined for E. coli strains comprisingdifferent E. coli mar genes having mutations in the firsthelix-turn-helix domain. The antibiotic MICs are shown in Table 5. TheA10 strain has an (Ala Ser Arg4 Ala Ser) inseration at amino acid 31.The A12 strain has a substitution of Val to Ala at position 33. The B7strain has an AlsSerAla5Ser insertion at amino acid position 40. The B9strain has a Trp to Ala substitution at position 42 and a His to Sersubstitution at position 43. The Lys14 strain has a Lys to Glnsubstitution at position 41. The Trp 15 strain has a Trp to Alasubstitution at position 42. The Phe21 strain has a Phe to Leusubstitution at position 48. The Ser2Leu3 strain has a Ser to Alasubstitution at position 29 and a Leu to Ala substitution at position30. The Ser2Ala strain has a Ser to Ala substitution at position 29.

Example 8 Mutations in the Second Alpha Helix of the FirstHelix-Turn-Helix Domain of MarA

[0170] To determine the roles of the MarA HTH domains in more detail,additional mutants in these regions were devised. These mutants weretested for their ability to promote a Mar phenotype in E. coli and forchanges in their affinity for the mar promoter. The mutants weresynthesized in amounts comparable to the wild type MarA as determined byWestern blot analysis. These mutants were defective in their ability topromote drug resistance, and exhibited lowered DNA binding affinities.Changes within the first alpha helix of the first HTH domain were lessdetrimental to MarA function than mutations in the second alpha helix ofthe first HTH domain.

Example 9 Design of Oligomers to Create Mutations in the SecondHelix-Turn-Helix Domain of MarA

[0171] As was done for the first helix-turn-helix domain of MarA,mutagenic oligomers can be designed to make mutations in the secondhelix-turn-helix domain of MarA. Exemplary oligomers for mutating thisregion of MarA are illustrated in FIG. 4.

[0172] Equivalents

[0173] Those skilled in the art will recognize, or be able to ascertainusing no more than routine experimentation, numerous equivalents to thespecific polypeptides, nucleic acids, methods, assays and reagentsdescribed herein. Such equivalents are considered to be within the scopeof this invention and are covered by the following claims. TABLE 1 SomeBacterial MarA homologs^(a) Gram-negative Gram-positive bacteriabacteria Escherichia coli Lactobacillus helveticus MarA (1) U34257 (39)OrfR (2, 3) Azorhizobium caulinodans SoxS (4, 5) S52856 (40) AfrR (6)Streptomyces spp. AraC (7) U21191 (41) CelD (8) AraL (42) D90812 (9)Streptococcus mutans FapR (10, 11) MsmR (43) MelR (12) Pediococcuspentosaceus ORF _f375 (13, 14 RafR (44) RhaR (15, 16, 17) Photobacteriumleiognathi RhaS (18) LumQ (45) Rob (19) Bacillus subtilis U73857 (20)AdaA (46) XylR (21) YbbB (47) YijO (22) YfiF (48) Proteus vulgaris YisR(49) PqrA (23) YzbC (50) Salmonella typhimurium MarA (24) InvF (25) PocR(26) Kiebsiella pneumoniae RamA (27) Haemophilus influenzae Ya52 (28)Yersinia spp. CafR (29) LcrF (30) or VirF (30) Providencia stuartii AarP(31) Pseudomonas spp. MmsR (32) TmbS (33) Xy1S (34) Xys1, 2, 3, 4 (35,36) Cyanobacteria Synechocystis spp. LumQ (38) PchR (38)

[0174] References for Table 1:

[0175] (1) S. P. Cohen, et al. 1993. J. Bacteriol. 175:1484-1492

[0176] (2) G. M. Braus, et al. 1984. J. Bacteriol. 160:504-509

[0177] (3) K. Schollmeier, et al., 1984. J. Bacteriol. 160:499-503

[0178] (4) C. F. Amabile-Cuevas, et al., 1991. Nucleic Acids Res.19:4479-4484

[0179] (5) J. Wu, et al., 1991. J. Bacteriol. 173:2864-2871

[0180] (6) M. K. Wolf, et al., 1990. Infect. Immun. 58:1124-1128

[0181] (7) C. M. Stoner, et al. 1982. J. Mol. Biol. 153:649-652

[0182] (8) L. L. Parker, et al., 1990. Genetics 123:455-471

[0183] (9) H. Mori, 1996. Unpublished data taken from the NCBI databases(10) P. Klaasen, et al., 1990. Mol. Microbiol. 4:1779-1783

[0184] (11) M. Ahmed, et al., 1994. J. Biol. Chjem 269-28506-28513

[0185] (12) C. Webster, et al., 1989. Gene 83:207-213

[0186] (13) G. Plunkett, III. 1995. Unpublished (14) C Garcia-Martin, etal., 1992. J. Gen. Microbiol. 138:1109-1116

[0187] (15) G. Plunkett, III., et al. 1993. Nucleic Acids Res.21:3391-3398

[0188] (16) C. G. Tate, et al. 1992. J. Biol. Chem. 267:6923-6932

[0189] (17) J. F. Tobin et al., 1987. J. Mol. Biol. 196:789-799

[0190] (18) J. Nishitani, 1991. Gene 105:37-42

[0191] (19) R. E. Benz, et al., 1993. Zentralbl. Bakteriol. Parasitenkd.Infektionskr. Hyg. Abt.

[0192] (20) 1 Orig. 278:187-196

[0193] (21) M. Duncan, et al., 1996. Unpublished data (22) H. J. Sofia,et al., 1994. Nucleic Acids Res. 22:2576-2586

[0194] (23) F. R. Blattner, et al., 1993. Nucleic Acids Res.21:5408-5417

[0195] (24) H. Ishida, et al., 1995. Antimicrob. Agents Chemother.39:453-457

[0196] (25) M. C. Sulavik, et al., 1997. J. Bacteriol. 179:1857-1866

[0197] (26) K. Kaniga, et al., 1994. Mol. Microbiol. 13:555-568

[0198] (27) J. R. Roth, et al. 1993. J. Bacteriol. 175:3303-3316

[0199] (28) A. M. George, et al., 1983. J. Bacteriol. 155:541-548

[0200] (29) R. D. Fleischmann, et al., 1995. Science 269:469-512

[0201] (30) E. E. Galyov, et al., 1991. FEBS Lett. 286:79-82 (31) N. P.Hoe, et al., 1992. J. Bacteriol. 174:4275-4286

[0202] (32) G. Cornelis, et al., 1989. J. Bacteriol. 171:254-262

[0203] (33) D. R. Macinga, et al., 1995. J. Bacteriol. 177:3407-3413

[0204] (34) M. I. Steele, et al., 1992. J. Biol. Chem. 267:13585-13592

[0205] (35) G. Deho, et al., 1995. Unpublished data (36) N. Mermod, etal., 1984. EMBO J. 3:2461-2466

[0206] (37) S. J. Assinder, et al., 1992. Nucleic Acids Res. 20:5476

[0207] (38) S. J. Assinder, et al., 1993. J. Gen. Microbiol. 139:557-568

[0208] (39) E. G. Dudley, et al., 1996. J. Bacteriol. 178:701-704

[0209] (40) D. Geelen, et al., 1995. Unpublished data (41) J. Kormanec,et al., 1995. Gene 165:77-80

[0210] (42) C. W. Chen, et al., 1992. J. Bacteriol. 174:7762-7769

[0211] (43) R. R. Russell, et al., 1992. J. Biol. Chem, 267:4631-4637

[0212] (44) K. K. Leenhouts, et al., 1995. Unpublished data (45) J. W.Lin, et al., 1995. Biochem. Biophys. Res. Commun. 217:684-695

[0213] (46) F. Morohoshi, et al. 1990. Nucleic Acids Res. 18:5473-5480

[0214] (47) M. Rosenberg, et al., 1979. Annu. Rev. Genet. 13:319-353

[0215] (48) H. Yamamoto, et al., 1996. Microbiology 142:1417-1421

[0216] (49) L. B. Bussey, et al., 1993. J. Bacteriol. 175:6348-6353

[0217] (50) P. G. Quirk, et al., 1994. Bichim. Biophys. Acta 1186:27-34TABLE 2 Bacterial strains and plasmids used in the Examples Strain orplasmid Description Reference or Source Strain E. coli AG100 Wild typeJ. Bacteriol., 1983 155: 531-540 E. coli AG102 Mar mutant of AG100 J.Bacteriol., 1983 155: 531-540 E. coli BL21 Expression strain for pETNovagen vectors M. smegmatis Electroporation Mol. Microbiol, MC²155competent 1990, 4: 1911-1919 Plasmids pMV261 Mycobacterium-E. colishuttle Nature, 1991, vector 351: 456-460 pPM10 pMV261::marA This studypPM10R pMV261::marA in antisense This study orientation pPM11pMV261::marR This study pPM1989R pPM10 insertional mutant This study ofhelix A (FIG. 1) pPM2016A pPM10 insertional mutant This study of helix B(FIG. 1) pET13a T7 expression vector Methods Enzymol, 1990, 185: 60-89pEC10 pET13::marA This study pEC1989R pEC10 insertional mutuant Thisstudy of helix A (FIG. 1) pEC2016A pEC10 insertional mutuant This studyof helix B (FIG. 1)

[0218] TABLE 3 Antibiotic susceptibilities of M. smegmatis mc²155microbes with and without plasmids bearing different E. coli mar genes %Growth in antibiotic gradient^(a) M. smegmatis RIF INH CML TET ETMmicrobe 30° C. 37° C. 30° C. 37° C. 30° C. 37° C. 30° C. 37° C. 30° C.37° C. Wild type 11 ± 1.2 11 ± 1.1 21 ± 1.2 20 ± 1.0 20 ± 0.8 22 ± 0.920 ± 1.9 19 ± 12  40 ± 2.2 44 ± 2.1 Transformants bearing plasmids:pMV261 22 ± 1.1 45 ± 2.6 22 ± 1.3 20 ± 1.0 22 ± 0.9 23 ± 1.1 20 ± 1.8 20± 1.3 42 ± 2.4 44 ± 1.5 pPM10 68 ± 1.6 90 ± 2.1 15 ± 1.5 45 ± 2.2 13 ±1.1 46 ± 1.6 15 ± 2.1 95 ± 3.1 38 ± 1.8 63 ± 2.7 pPM10R 24 ± 1.0 47 ±2.2 20 ± 0.9 24 ± 1.7 22 ± 0.9 27 ± 1.1 21 ± 2.0 20 ± 1.0 38 ± 2.0 45 ±2.4 pPM11 24 ± 1.0 45 ± 2.1 22 ± 1.0 23 ± 1.2 22 ± 1.0 27 ± 1.0 22 ± 2.220 ± 1.3 40 ± 2.0 46 ± 2.4 pPM1989R ND 10 ± 1.0 ND 18 ± 2.1 ND 26 ± 1.4ND 20 ± 2.0 ND 48 ± 2.2 pPM2016A ND 13 ± 0.8 ND 22 ± 1.0 ND 27 ± 2.2 ND22 ± 1.7 ND 50 ± 2.4

[0219] TABLE 4 Antibiotic susceptibilities of E. coli microbes withplasmids bearing different E. coli mar genes Plasmid borne by % Gowth inantibiotic gradient^(a) E. coli transformant CML TET AMP NAL pET13a 20 ±1.1 33 ± 1.0 2 ± 1.1 17 ± 0.8 pEC10 100 100 11 ± 2.0  100 pEC1989R 20 ±1.6 33 ± 1.4 2 ± 1.0 17 ± 1.2 pEC2016A 20 ± 1.3 33 ± 1.4 2 ± 1.7 17 ±1.1

[0220] TABLE 5 Resistance Pattern of MarA Mutants in 1stHelix-Turn-Helix Domain Strain Tet Chlor Nal Ac Rif Cipro Nor Amp GentaCeph A₁₀Ala₁₄ 0.75 ± .25 1.0 0.38 ± .12 10.7 ± 2.3 <0.002 <0.016 0.74 ±.02 1.0 1.5 Ser₂Ala 1.25 ± .43  3.3 ± .57  1.1 ± .38 >32 <0.002 <0.016 2.4 ± .27 1.0 3.0 pET13A 0.71 ± .31 1.0 0.23 ± .03 13.3 ± 4.6 <0.002<0.016 0.76 ± .11 0.75 1.5 A10 0.75 ± .25 1.08 ± .38 0.38 ± .13 12 ± 4<0.002 <0.016  0.8 ± .07 0.75 1.5 A12 0.75 ± .25  1.5 ± 0.5 0.54 ± .18 16 <0.002 <0.016  1.3 ± 0.18 0.75 0.094 B7 0.58 ± .14  1.3 ± .28 0.33 ±.14 17.3 ± 6.1 <0.002 <0.016  0.9 ± .24 0.75 3 B9 0.92 ± .52  1.2 ± .280.42 ± .06   15 ± 2.3 <0.002 <0.016 0.84 ± .26 4 1.5 Lys14 0.67 ± .14 1.2 ± .28 0.38 ± .12 13.3 ± 2.3 <0.002 <0.016  0.9 ± .23 3 2 Ser2Leu30.75 1.0 0.33 ± .07 14.6 ± 2.3 <0.002 <0.016 0.75 ± .03 0.75 1.5 Trp150.58 ± .14 0.83 ± .28 0.25 ± .12 10.6 ± 2.3 <0.002 <0.016 0.84 ± .050.75 3 A10Ala 6 0.92 ± .52 0.83 ± .14 0.37 ± .12 12 ± 4 <0.002 <0.0160.75 ± .03 0.75 1.5 Phe21 0.66 ± .14 0.92 ± .14 0.33 ± .14 10.6 ± 2.3<0.002 <0.016 0.78 ± .04 1.0 2 MarA  1.2 ± .28  2.5 ± .86 0.96 ± .56 >32<0.002 <0.016  3.0 ± .38 1.0 3 BL21 0.75 0.66 ± .14 0.23 ± .03  6.7 ±1.1 <0.002 <0.016 <0.125 1.0 1.5

[0221]

1 216 1 7878 DNA Escherichia coli CDS (1894)...(2283) 1 gttaactgtggtggttgtca ccgcccatta cacggcatac agctatatcg agccttttgt 60 acaaaacattgcgggattca gcgccaactt tgccacggca ttactgttat tactcggtgg 120 tgcgggcattattggcagcg tgattttcgg taaactgggt aatcagtatg cgtctgcgtt 180 ggtgagtacggcgattgcgc tgttgctggt gtgcctggca ttgctgttac ctgcggcgaa 240 cagtgaaatacacctcgggg tgctgagtat tttctggggg atcgcgatga tgatcatcgg 300 gcttggtatgcaggttaaag tgctggcgct ggcaccagat gctaccgacg tcgcgatggc 360 gctattctccggcatattta atattggaat cggggcgggt gcgttggtag gtaatcaggt 420 gagtttgcactggtcaatgt cgatgattgg ttatgtgggc gcggtgcctg cttttgccgc 480 gttaatttggtcaatcatta tatttcgccg ctggccagtg acactcgaag aacagacgca 540 atagttgaaaggcccattcg ggcctttttt aatggtacgt tttaatgatt tccaggatgc 600 cgttaataataaactgcaca cccatacata ccagcaggaa tcccatcaga cgggagatcg 660 cttcaatgccacccttgccc accagccgca taattgcgcc ggagctgcgt aggcttcccc 720 acaaaataaccgccaccagg aaaaagatca gcggcggcgc aaccatcagt acccaatcag 780 cgaaggttgaactctgacgc actgtggacg ccgagctaat aatcatcgct atggttcccg 840 gaccggcagtacttggcatt gccagcggca caaaggcaat attggcactg ggttcatctt 900 ccagctcttccgacttgctt ttcgcctccg gtgaatcaat cgctttctgt tgcggaaaga 960 gcatccgaaaaccgataaac gcgacgatta agccgcctgc aattcgcaga ccgggaatcg 1020 aaatgccaaatgtatccatc accagttgcc cggcgtaata cgccaccatc atgatggcaa 1080 atacgtacaccgaggccatc aacgactgac gattacgttc ggcactgttc atgttgcctg 1140 ccaggccaagaaataacgcg acagttgtta atgggttagc taacggcagc aacaccacca 1200 gccccaggccaattgcttta aacaaatcta acattggtgg ttgttatcct gtgtatctgg 1260 gttatcagcgaaaagtataa ggggtaaaca aggataaagt gtcactcttt agctagcctt 1320 gcatcgcattgaacaaaact tgaaccgatt tagcaaaacg tggcatcggt caattcattc 1380 atttgacttatacttgcctg ggcaatatta tcccctgcaa ctaattactt gccagggcaa 1440 ctaatgtgaaaagtaccagc gatctgttca atgaaattat tccattgggt cgcttaatcc 1500 atatggttaatcagaagaaa gatcgcctgc ttaacgagta tctgtctccg ctggatatta 1560 ccgcggcacagtttaaggtg ctctgctcta tccgctgcgc ggcgtgtatt actccggttg 1620 aactgaaaaaggtattgtcg gtcgacctgg gagcactgac ccgtatgctg gatcgcctgg 1680 tctgtaaaggctgggtggaa aggttgccga acccgaatga caagcgcggc gtactggtaa 1740 aacttaccaccggcggcgcg gcaatatgtg aacaatgcca tcaattagtt ggccaggacc 1800 tgcaccaagaattaacaaaa aacctgacgg cggacgaagt ggcaacactt gagtatttgc 1860 ttaagaaagtcctgccgtaa acaaaaaaga ggt atg acg atg tcc aga cgc aat 1914 Met Thr MetSer Arg Arg Asn 1 5 act gac gct att acc att cat agc att ttg gac tgg atcgag gac aac 1962 Thr Asp Ala Ile Thr Ile His Ser Ile Leu Asp Trp Ile GluAsp Asn 10 15 20 ctg gaa tcg cca ctg tca ctg gag aaa gtg tca gag cgt tcgggt tac 2010 Leu Glu Ser Pro Leu Ser Leu Glu Lys Val Ser Glu Arg Ser GlyTyr 25 30 35 tcc aaa tgg cac ctg caa cgg atg ttt aaa aaa gaa acc ggt cattca 2058 Ser Lys Trp His Leu Gln Arg Met Phe Lys Lys Glu Thr Gly His Ser40 45 50 55 tta ggc caa tac atc cgc agc cgt aag atg acg gaa atc gcg caaaag 2106 Leu Gly Gln Tyr Ile Arg Ser Arg Lys Met Thr Glu Ile Ala Gln Lys60 65 70 ctg aag gaa agt aac gag ccg ata ctc tat ctg gca gaa cga tat ggc2154 Leu Lys Glu Ser Asn Glu Pro Ile Leu Tyr Leu Ala Glu Arg Tyr Gly 7580 85 ttc gag tcg caa caa act ctg acc cga acc ttc aaa aat tac ttt gat2202 Phe Glu Ser Gln Gln Thr Leu Thr Arg Thr Phe Lys Asn Tyr Phe Asp 9095 100 gtt ccg ccg cat aaa tac cgg atg acc aat atg cag ggc gaa tcg cgc2250 Val Pro Pro His Lys Tyr Arg Met Thr Asn Met Gln Gly Glu Ser Arg 105110 115 ttt tta cat cca tta aat cat tac aac agc tag ttgaaaacgtgacaacgtca 2303 Phe Leu His Pro Leu Asn His Tyr Asn Ser * 120 125ctgaggcaat catgaaacca ctttcatccg caatagcagc tgcgcttatt ctcttttccg 2363cgcagggcgt tgcggaacaa accacgcagc cagttgttac ttcttgtgcc aatgtcgtgg 2423ttgttccccc atcgcaggaa cacccaccgt ttgatttaaa tcacatgggt actggcagtg 2483ataagtcgga tgcgctcggc gtgccctatt ataatcaaca cgctatgtag tttgttctgg 2543ccccgacatc tcggggctta ttaacttccc acctttaccg ctttacgcca ccgcaagcca 2603aatacattga tatacagccc ggtcataatg agcaccgcac ctaaaaattg cagacccgtt 2663aagcgttcat ccaacaatag tgccgcactt gccagtccta ctacgggcac cagtaacgat 2723aacggtgcaa cccgccaggt ttcatagcgt cccagtaacg tcccccagat cccataacca 2783acaattgtcg ccacaaacgc cagatacatc agagacaaga tggtggtcat atcgatagta 2843accagactgt gaatcatggt tgcggaacca tcgagaatca gcgaggcaac aaagaaggga 2903atgattggga ttaaagcgct ccagattacc agcgacatca ccgccggacg cgttgagtgc 2963gacatgatct ttttattgaa gatgttgcca cacgcccaac taaatgctgc cgccagggtc 3023aacataaagc cgagcatcgc cacatgctga ccgttcagac tatcttcgat taacaccagt 3083acgccaaaaa tcgctaaggc gatccccgcc aattgtttgc catgcagtcg ctccccgaaa 3143gtaaacgcgc caagcatgat agtaaaaaac gcctgtgcct gtaacaccag cgaagccagt 3203ccagcaggca taccgaagtt aatggcacaa aaaagaaaag caaactgcgc aaaactgatg 3263gttaatccat accccagcag caaattcagt ggtactttcg gtcgtgcgac aaaaaagata 3323gccggaaaag cgaccagcat aaagcgcaaa ccggccagca tcagcgtggc atgttatgaa 3383gccccacttt gatgaccaca aaatttagcc cccatacgac cactaccagt agcgccaaca 3443ccccatcttt tcgcgacatt ctaccgcctc tgaatttcat cttttgtaag caatcaactt 3503agctgaattt acttttcttt aacagttgat tcgttagtcg ccggttacga cggcattaat 3563gcgcaaataa gtcgctatac ttcggatttt tgccatgcta tttctttaca tctctaaaac 3623aaaacataac gaaacgcact gccggacaga caaatgaact tatccctacg acgctctacc 3683agcgcccttc ttgcctcgtc gttgttatta accatcggac gcggcgctac cgtgccattt 3743atgaccattt acttgagtcg ccagtacagc ctgagtgtcg atctaatcgg ttatgcgatg 3803acaattgcgc tcactattgg cgtcgttttt agcctcggtt ttggtatcct ggcggataag 3863ttcgacaaga aacgctatat gttactggca attaccgcct tcgccagcgg ttttattgcc 3923attactttag tgaataacgt gacgctggtt gtgctctttt ttgccctcat taactgcgcc 3983tattctgttt ttgctaccgt gctgaaagcc tggtttgccg acaatctttc gtccaccagc 4043aaaacgaaaa tcttctcaat caactacacc atgctaaaca ttggctgacc atcggtccgc 4103cgctcggcac gctgttggta atgcagagca tcaatctgcc cttctggctg gcagctatct 4163gttccgcgtt tcccatgctt ttcattcaaa tttgggtaaa gcgcagcgag aaaatcatcg 4223ccacggaaac aggcagtgtc tggtcgccga aagttttatt acaagataaa gcactgttgt 4283ggtttacctg ctctggtttt ctggcttctt ttgtaagcgg cgcatttgct tcatgcattt 4343cacaatatgt gatggtgatt gctgatgggg attttgccga aaaggtggtc gcggttgttc 4403ttccggtgaa tgctgccatg gtggttacgt tgcaatattc cgtgggccgc cgacttaacc 4463cggctaacat ccgcgcgctg atgacagcag gcaccctctg tttcgtcatc ggtctggtcg 4523gttttatttt ttccggcaac agcctgctat tgtggggtat gtcagctgcg gtatttactg 4583tcggtgaaat catttatgcg ccgggcgagt atatgttgat tgaccatatt gcgccgccag 4643aaatgaaagc cagctatttt tccgcccagt ctttaggctg gcttggtgcc gcgattaacc 4703cattagtgag tggcgtagtg ctaaccagcc tgccgccttc ctcgctgttt gtcatcttag 4763cgttggtgat cattgctgcg tgggtgctga tgttaaaagg gattcgagca agaccgtggg 4823ggcagcccgc gctttgttga tttaagtcga acacaataaa gatttaattc agccttcgtt 4883taggttacct ctgctaatat ctttctcatt gagatgaaaa ttaaggtaag cgaggaaaca 4943caccacacca taaacggagg caaataatgc tgggtaatat gaatgttttt atggccgtac 5003tgggaataat tttattttct ggttttctgg ccgcgtattt cagccacaaa tgggatgact 5063aatgaacgga gataatccct cacctaaccg gccccttgtt acagttgtgt acaaggggcc 5123tgatttttat gacggcgaaa aaaaaccgcc agtaaaccgg cggtgaatgc ttgcatggat 5183agatttgtgt tttgctttta cgctaacagg cattttcctg cactgataac gaatcgttga 5243cacagtagca tcagttttct caatgaatgt taaacggagc ttaaactcgg ttaatcacat 5303tttgttcgtc aataaacatg cagcgatttc ttccggtttg cttaccctca tacattgccc 5363ggtccgctct tccaatgacc acatccagag gctcttcagg aaatgcgcga ctcacacctg 5423ctgtcacggt aatgttgata tgcccttcag aatgtgtgat ggcatggtta tcgactaact 5483ggcaaattct gacacctgca cgacatgctt cttcatcatt agccgctttg acaataatga 5543taaattcttc gcccccgtag cgataaaccg tttcgtaatc acgcgtccaa ctggctaagt 5603aagttgccag ggtgcgtaat actacatcgc cgattaaatg cccgtagtat cattaaccaa 5663tttaaatcgg tcaatatcca acaacattaa ataaagattc agaggctcag cgttgcgtaa 5723ctgatgatca aaggattcat caagaacccg acgacccggc aatcccgtca aaacatccat 5783attgctacgg atcgtcagca aataaatttt gtaatcggtt aatgccgcag taaaagaaag 5843caacccctcc tgaaaggcgt cgaaatgcgc gtcctgccag tgattttcaa caatagccag 5903cattaattcc cgaccacagt tatgcatatg ttgatgggca gaatccatta gccgaacgta 5963aggtaattca tcgttatcga gtggccccag atgatcaatc caccgaccaa actggcacag 6023tccataagaa tggttatccg ttatttctgg cttactggca tctctcgcga ccacgctgtg 6083aaacatactc accagccact ggtagtgggc atcgatagcc ttattgagat ttaacaagat 6143ggcatcaatt tccgttgtct tcttgatcat tgccactcct ttttcacagt tccttgtgcg 6203cgctattcta acgagagaaa agcaaaatta cgtcaatatt ttcatagaaa tccgaagtta 6263tgagtcatct ctgagataac attgtgattt aaaacaaaat cagcggataa aaaagtgttt 6323aattctgtaa attacctctg cattatcgta aataaaagga tgacaaatag cataacccaa 6383taccctaatg gcccagtagt tcaggccatc aggctaattt atttttattt ctgcaaatga 6443gtgacccgaa cgacggccgg cgcgcttttc ttatccagac tgccactaat gttgatcatc 6503tggtccggct gaacttctcg tccatcaaag acggccgcag gaataacgac attaatttca 6563ccgctcttat cgcgaaaaac gtaacggtcc tctcctttgt gagaaatcaa attaccgcgt 6623agtgaaaccg aagcgccatc gtgcatggtt tttgcgaaat caacggtcat tttttttgca 6683tcatcggttc cgcgatagcc atcttctatt gcatgaggcg gcggtggcgc tgcatcctgt 6743tttaaaccgc cctggtcatc tgccaacgca taaggcatga caagaaaact tgctaataca 6803atggcctgaa atttcatact aactccttaa ttgcgtttgg tttgacttat taagtctggt 6863tgctattttt ataattgcca aataagaata ttgccaattg ttataaggca tttaaaatca 6923gccaactagc tgtcaaatat acagagaatt taactcacta aagttaagaa gattgaaaag 6983tcttaaacat attttcagaa taatcggatt tatatgtttg aaaattatta tattggacga 7043gcatacagaa aaagcaaatc acctttacat ataaaagcgt ggacaaaaaa cagtgaacat 7103taatagagat aaaattgtac aacttgtaga taccgatact attgaaaacc tgacatccgc 7163gttgagtcaa agacttatcg cggatcaatt acgcttaact accgccgaat catgcaccgg 7223cggtaagttg gctagcgccc tgtgtgcagc tgaagataca cccaaatttt acggtgcagg 7283ctttgttact ttcaccgatc aggcaaagat gaaaatcctc agcgtaagcc agcaatctct 7343tgaacgatat tctgcggtga gtgagaaagt ggcagcagaa atggcaaccg gtgccataga 7403gcgtgcggat gctgatgtca gtattgccat taccggctac ggcggaccgg agggcggtga 7463agatggtacg ccagcgggta ccgtctggtt tgcgtggcat attaaaggcc agaactacac 7523tgcggttatg cattttgctg gcgactgcga aacggtatta gctttagcgg tgaggtttgc 7583cctcgcccag ctgctgcaat tactgctata accaggctgg cctggcgata tctcaggcca 7643gccattggtg gtgtttatat gttcaagcca cgatgttgca gcatcggcat aatcttaggt 7703gccttaccgc gccattgtcg atacaggcgt tccagatctt cgctgttacc tctggaaagg 7763atcgcctcgc gaaaacgcag cccattttca cgcgttaatc cgccctgctc aacaaaccac 7823tgataaccat catcggccaa catttgcgtc cacagataag cgtaataacc tgcag 7878 2 129PRT Escherichia coli 2 Met Thr Met Ser Arg Arg Asn Thr Asp Ala Ile ThrIle His Ser Ile 1 5 10 15 Leu Asp Trp Ile Glu Asp Asn Leu Glu Ser ProLeu Ser Leu Glu Lys 20 25 30 Val Ser Glu Arg Ser Gly Tyr Ser Lys Trp HisLeu Gln Arg Met Phe 35 40 45 Lys Lys Glu Thr Gly His Ser Leu Gly Gln TyrIle Arg Ser Arg Lys 50 55 60 Met Thr Glu Ile Ala Gln Lys Leu Lys Glu SerAsn Glu Pro Ile Leu 65 70 75 80 Tyr Leu Ala Glu Arg Tyr Gly Phe Glu SerGln Gln Thr Leu Thr Arg 85 90 95 Thr Phe Lys Asn Tyr Phe Asp Val Pro ProHis Lys Tyr Arg Met Thr 100 105 110 Asn Met Gln Gly Glu Ser Arg Phe LeuHis Pro Leu Asn His Tyr Asn 115 120 125 Ser 3 20 PRT Artificial Sequenceconsensus sequence 3 Xaa Xaa Xaa Xaa Ala Xaa Xaa Xaa Xaa Xaa Ser Xaa XaaXaa Leu Xaa 1 5 10 15 Xaa Xaa Phe Xaa 20 4 22 PRT Artificial Sequenceconsensus sequence 4 Xaa Xaa Ile Xaa Xaa Ile Ala Xaa Xaa Xaa Gly Phe XaaSer Xaa Xaa 1 5 10 15 Xaa Phe Xaa Xaa Xaa Xaa 20 5 22 PRT ArtificialSequence consensus sequence 5 Xaa Xaa Xaa Ala Xaa Xaa Xaa Gly Xaa SerXaa Xaa Xaa Leu Gln Xaa 1 5 10 15 Xaa Phe Xaa Xaa Xaa Xaa 20 6 23 PRTArtificial Sequence consensus sequence 6 Ile Xaa Asp Ile Ala Xaa Xaa XaaGly Phe Xaa Ser Xaa Xaa Phe Xaa 1 5 10 15 Xaa Xaa Phe Xaa Xaa Xaa Xaa 207 18 DNA Artificial Sequence consensus sequence 7 ctagcgcggc ggcggcgg 188 18 DNA Artificial Sequence primer 8 gcgccgccgc cgccgatc 18 9 8 PRTArtificial Sequence consensus sequence 9 Ala Ser Arg Arg Arg Arg Ala Ser1 5 10 18 PRT Escherichia coli 10 Pro Leu Ser Leu Glu Lys Val Ser GluArg Ser Gly Tyr Ser Lys Trp 1 5 10 15 His Leu 11 8 PRT ArtificialSequence consensus sequence 11 Ala Ser Ala Ala Ala Ala Ala Ser 1 5 12 47PRT Escherichia coli 12 Leu Glu Lys Val Ser Glu Arg Ser Gly Tyr Ser LysTrp His Leu Gln 1 5 10 15 Arg Met Phe Lys Lys Glu Thr Gly His Ser LeuGly Gln Tyr Ile Arg 20 25 30 Ser Arg Lys Met Thr Glu Ile Ala Gln Lys LeuLys Glu Ser Asn 35 40 45 13 47 PRT Salmonella typhimurium 13 Leu Asp AlaPhe Cys Gln Gln Glu Gln Cys Ser Glu Arg Val Leu Arg 1 5 10 15 Ala GlnPhe Arg Ala Gln Thr Gly Met Thr Ile Asn Gln Tyr Leu Arg 20 25 30 Gln ValArg Ile Cys His Ala Gln Tyr Leu Leu Gln His Ser Pro 35 40 45 14 47 PRTEscherichia coli 14 Leu Asp Lys Phe Cys Asp Glu Ala Ser Cys Ser Glu ArgVal Leu Arg 1 5 10 15 Gln Gln Phe Arg Gln Gln Thr Gly Met Thr Ile AsnGln Tyr Leu Arg 20 25 30 Gln Val Arg Ile Cys His Ala Gln Tyr Leu Leu GlnHis Ser Pro 35 40 45 15 47 PRT Escherichia coli 15 Trp Asp Ala Val AlaAsp Gln Phe Ser Leu Ser Leu Arg Thr Leu His 1 5 10 15 Arg Gln Leu LysGln Gln Thr Gly Leu Thr Pro Gln Arg Tyr Leu Asn 20 25 30 Arg Leu Arg LeuMet Lys Ala Arg His Leu Leu Arg His Ser Glu 35 40 45 16 47 PRTSalmonella typhimurium 16 Trp Glu Ala Val Ala Glu Gln Phe Ser Leu SerLeu Arg Thr Leu His 1 5 10 15 Arg Gln Leu Lys Gln Gln Thr Gly Leu ThrPro Gln Arg Tyr Leu Asn 20 25 30 Arg Leu Arg Leu Ile Lys Ala Arg His LeuLeu Arg His Ser Asp 35 40 45 17 47 PRT Pseudomonas aeroginosa 17 Leu SerAsp Phe Ser Arg Glu Phe Gly Met Gly Leu Thr Thr Phe Lys 1 5 10 15 GluLeu Phe Gly Ser Val Tyr Gly Val Ser Pro Arg Ala Trp Ile Ser 20 25 30 GluArg Arg Ile Leu Tyr Ala His Gln Leu Leu Leu Asn Ser Asp 35 40 45 18 47PRT Yersinia pestis 18 Leu Ser Lys Phe Ala Arg Glu Phe Gly Met Gly LeuThr Thr Phe Lys 1 5 10 15 Glu Leu Phe Gly Thr Val Tyr Gly Ile Ser ProArg Ala Trp Ile Ser 20 25 30 Glu Arg Arg Ile Leu Tyr Ala His Gln Leu LeuLeu Asn Gly Lys 35 40 45 19 47 PRT Yersinia enterocolitica 19 Leu SerLys Phe Ala Arg Glu Phe Gly Met Gly Leu Thr Thr Phe Lys 1 5 10 15 GluLeu Phe Gly Thr Val Tyr Gly Ile Ser Pro Arg Ala Trp Ile Ser 20 25 30 GluArg Arg Ile Leu Tyr Ala His Gln Leu Leu Leu Asn Gly Lys 35 40 45 20 47PRT Citrobacter freundii 20 Ile Ala Ser Val Ala Gln His Val Cys Leu SerPro Ser Arg Leu Ser 1 5 10 15 His Leu Phe Arg Gln Gln Leu Gly Ile SerVal Leu Ser Trp Arg Glu 20 25 30 Asp Gln Arg Ile Ser Gln Ala Lys Leu LeuLeu Ser Thr Thr Arg 35 40 45 21 47 PRT Escherichia coli 21 Ile Ala SerVal Ala Gln His Val Cys Leu Ser Pro Ser Arg Leu Ser 1 5 10 15 His LeuPhe Arg Gln Gln Leu Gly Ile Ser Val Leu Ser Trp Arg Glu 20 25 30 Asp GlnArg Ile Ser Gln Ala Lys Leu Leu Leu Ser Thr Thr Arg 35 40 45 22 47 PRTEscherichia coli 22 Ile Ala Ser Val Ala Gln His Val Cys Leu Ser Pro SerArg Leu Ser 1 5 10 15 His Leu Phe Arg Gln Gln Leu Gly Ile Ser Val LeuSer Trp Arg Glu 20 25 30 Asp Gln Arg Ile Ser Gln Ala Lys Leu Leu Leu SerThr Thr Arg 35 40 45 23 47 PRT Erwinia cloacae 23 Ile Asp Glu Val AlaArg His Val Cys Leu Ser Pro Ser Arg Leu Ala 1 5 10 15 His Leu Phe ArgGlu Gln Val Gly Ile Asn Ile Leu Arg Trp Arg Glu 20 25 30 Asp Gln Arg ValIle Arg Ala Lys Leu Leu Leu Gln Thr Thr Gln 35 40 45 24 47 PRTSalmonella typhimurium 24 Leu Glu Asp Val Ala Ser His Val Tyr Leu SerPro Tyr Tyr Phe Ser 1 5 10 15 Lys Leu Phe Lys Lys Tyr Gln Gly Ile GlyPhe Asn Ala Trp Val Asn 20 25 30 Arg Gln Arg Met Val Ser Ala Arg Glu LeuLeu Cys His Ser Asp 35 40 45 25 47 PRT Escherichia coli 25 Arg Glu SerVal Ala Gln Ala Phe Tyr Ile Ser Pro Asn Tyr Leu Ser 1 5 10 15 His LeuPhe Gln Lys Thr Gly Ala Ile Gly Phe Asn Glu Tyr Leu Asn 20 25 30 His ThrArg Leu Glu His Ala Lys Thr Leu Leu Lys Gly Tyr Asp 35 40 45 26 47 PRTBacillus subtilis 26 Leu Ala Gln Leu Ser Gln Met Ala Gly Ile Ser Ala LysHis Tyr Ser 1 5 10 15 Glu Ser Phe Lys Lys Trp Thr Gly Gln Ser Val ThrGlu Phe Ile Thr 20 25 30 Lys Thr Arg Ile Thr Lys Ala Lys Arg Leu Met AlaLys Ser Asn 35 40 45 27 47 PRT Bacillus subtilis 27 Leu Thr Asp Val AlaSer His Phe His Ile Ser Gly Arg His Leu Ser 1 5 10 15 Arg Leu Phe AlaAla Glu Leu Gly Val Ser Tyr Ser Glu Phe Val Gln 20 25 30 Asn Glu Lys IleAsn Lys Ala Ala Glu Leu Leu Lys Ser Thr Asn 35 40 45 28 47 PRTPhotobacterium leiognathi 28 Val Ala Glu Leu Ser Ser Val Ala Phe Leu AlaGln Ser Gln Phe Tyr 1 5 10 15 Ala Leu Phe Lys Ser Gln Met Gly Ile ThrPro His Gln Tyr Val Leu 20 25 30 Arg Lys Arg Leu Asp Leu Ala Lys Gln LeuIle Ala Glu Arg Gln 35 40 45 29 47 PRT Streptomyces atratus 29 Val AlaGlu Leu Ala Ser Ala Ala Ala Val Ser Arg Ser Thr Leu Ala 1 5 10 15 AlaArg Phe Lys Ala Thr Val Gly Gln Gly Pro Leu Glu Tyr Leu Thr 20 25 30 ArgTrp Arg Ile Glu Leu Thr Ala Arg Gln Leu Arg Glu Gly Ser 35 40 45 30 47PRT Streptomyces limosus 30 Val Ala Glu Leu Ala Ser Ala Ala Ala Val SerArg Ser Thr Leu Ala 1 5 10 15 Ala Arg Phe Lys Ala Thr Val Gly Gln GlyPro Leu Glu Tyr Leu Thr 20 25 30 Arg Trp Arg Ile Glu Leu Thr Ala Arg GlnLeu Arg Glu Gly Asn 35 40 45 31 47 PRT Escherichia coli 31 Val Glu SerLeu Ala Ser Ile Ala His Met Ser Arg Ala Ser Phe Ala 1 5 10 15 Gln LeuPhe Arg Asp Val Ser Gly Thr Thr Pro Leu Ala Val Leu Thr 20 25 30 Lys LeuArg Leu Gln Ile Ala Ala Gln Met Phe Ser Arg Glu Thr 35 40 45 32 47 PRTHaemophilus influenzae 32 Ile Glu Gln Leu Ala Glu Leu Ala Thr Met SerArg Ala Asn Phe Ile 1 5 10 15 Arg Ile Phe Gln Gln His Ile Gly Met SerPro Gly Arg Phe Leu Thr 20 25 30 Lys Val Arg Leu Gln Ser Ala Ala Phe LeuLeu Lys Gln Ser Gln 35 40 45 33 47 PRT Pseudomonas aeroginosa 33 Leu GluArg Leu Ala Ala Phe Cys Asn Leu Ser Lys Phe His Phe Val 1 5 10 15 SerArg Tyr Lys Ala Ile Thr Gly Arg Thr Pro Ile Gln His Phe Leu 20 25 30 HisLeu Lys Ile Glu Tyr Ala Cys Gln Leu Leu Asp Ser Ser Asp 35 40 45 34 47PRT Streptococcus mutans 34 Val Asn Asp Ile Ala Lys Lys Leu Asn Leu SerArg Ser Tyr Leu Tyr 1 5 10 15 Lys Ile Phe Arg Lys Ser Thr Asn Leu SerIle Lys Glu Tyr Ile Leu 20 25 30 Gln Val Arg Met Lys Arg Ser Gln Tyr LeuLeu Glu Asn Pro Lys 35 40 45 35 47 PRT Pediococcus pentosaceus 35 IleMet Asp Leu Cys His Tyr Leu Asn Leu Ser Arg Ser Tyr Leu Tyr 1 5 10 15Thr Leu Phe Lys Thr His Ala Asn Thr Ser Pro Gln Lys Leu Leu Thr 20 25 30Lys Leu Arg Leu Glu Asp Ala Lys Gln Arg Leu Ser Thr Ser Asn 35 40 45 3647 PRT Escherichia coli 36 Val Ala Asp Met Ala Ala Thr Ile Pro Cys SerGlu Ala Trp Leu Arg 1 5 10 15 Arg Leu Phe Leu Arg Tyr Thr Gly Lys ThrPro Lys Glu Tyr Tyr Leu 20 25 30 Asp Ala Arg Leu Asp Leu Ala Leu Ser LeuLeu Lys Gln Gln Gly 35 40 45 37 46 PRT Pseudoalteromonas carrgenorora 37Ile Asp Thr Val Ala Phe Ser Val Gly Val Ser Arg Ser Tyr Leu Val 1 5 1015 Lys Gln Phe Lys Leu Ala Thr Asn Lys Thr Ile Asn Asn Arg Ile Ile 20 2530 Glu Val Arg Ile Glu Gln Ala Lys Lys Val Leu Leu Lys Lys 35 40 45 3847 PRT Escherichia coli 38 Val Asp Gln Val Leu Asp Ala Val Gly Ile SerArg Ser Asn Leu Glu 1 5 10 15 Lys Arg Phe Lys Glu Glu Val Gly Glu ThrIle His Ala Met Ile His 20 25 30 Ala Glu Lys Leu Glu Lys Ala Arg Ser LeuLeu Ile Ser Thr Thr 35 40 45 39 47 PRT Haemophilus influenzae 39 Val GlyGln Val Leu Asp His Leu Glu Thr Ser Arg Ser Asn Leu Glu 1 5 10 15 GlnArg Phe Lys Asn Glu Met Asn Lys Thr Ile His Gln Val Ile His 20 25 30 GluGlu Lys Ile Ser Arg Ala Lys Asn Leu Leu Gln Gln Thr Asp 35 40 45 40 47PRT Bacillus subtilis 40 Leu Glu Ser Leu Ala Asp Ile Cys His Gly Ser ProTyr His Met His 1 5 10 15 Arg Thr Phe Lys Lys Ile Lys Gly Ile Thr LeuVal Glu Tyr Ile Gln 20 25 30 Gln Val Arg Val His Ala Ala Lys Lys Tyr LeuIle Gln Thr Asn 35 40 45 41 47 PRT Escherichia coli 41 Leu Glu Asn MetVal Ala Leu Ser Ala Lys Ser Gln Glu Tyr Leu Thr 1 5 10 15 Arg Ala ThrGln Arg Tyr Tyr Gly Lys Thr Pro Met Gln Ile Ile Asn 20 25 30 Glu Ile ArgIle Asn Phe Ala Lys Lys Gln Leu Glu Met Thr Asn 35 40 45 42 47 PRTEscherichia coli 42 Ile Asn Asp Val Ala Glu His Val Lys Leu Asn Ala AsnTyr Ala Met 1 5 10 15 Gly Ile Phe Gln Arg Val Met Gln Leu Thr Met LysGln Tyr Ile Thr 20 25 30 Ala Met Arg Ile Asn His Val Arg Ala Leu Leu SerAsp Thr Asp 35 40 45 43 47 PRT Pseudomonas aeroginosa 43 Leu Asp Thr LeuAla Ser Arg Val Gly Met Asn Pro Arg Lys Leu Thr 1 5 10 15 Ala Gly PheArg Lys Val Phe Gly Ala Ser Val Phe Gly Tyr Leu Gln 20 25 30 Glu Tyr ArgLeu Arg Glu Ala His Arg Met Leu Cys Asp Glu Glu 35 40 45 44 47 PRTSalmonella typhimurium 44 Leu Glu Lys Val Ser Glu Arg Ser Gly Tyr SerLys Trp His Leu Gln 1 5 10 15 Arg Met Phe Lys Lys Glu Thr Gly His SerLeu Gly Gln Tyr Ile Arg 20 25 30 Ser Arg Lys Met Thr Glu Ile Ala Gln LysLeu Lys Glu Ser Asn 35 40 45 45 47 PRT Escherichia coli 45 Leu Glu LysVal Ser Glu Arg Ser Gly Tyr Ser Lys Trp His Leu Gln 1 5 10 15 Arg MetPhe Lys Lys Glu Thr Gly His Ser Leu Gly Gln Tyr Ile Arg 20 25 30 Ser ArgLys Met Thr Glu Ile Ala Gln Lys Leu Lys Glu Ser Asn 35 40 45 46 47 PRTProteus Inconstans 46 Ile Asp Thr Ile Ala Asn Lys Ser Gly Tyr Ser LysTrp His Leu Gln 1 5 10 15 Arg Ile Phe Lys Asp Phe Lys Gly Cys Thr LeuGly Glu Tyr Val Arg 20 25 30 Lys Arg Arg Leu Leu Glu Ala Ala Lys Ser LeuGln Glu Lys Asp 35 40 45 47 47 PRT Providencia stuartii 47 Leu Asp AspIle Ala Gln His Ser Gly Tyr Thr Lys Trp His Leu Gln 1 5 10 15 Arg ValPhe Arg Lys Ile Val Gly Met Pro Leu Gly Glu Tyr Ile Arg 20 25 30 Arg ArgArg Ile Cys Glu Ala Ala Lys Glu Leu Gln Thr Thr Asn 35 40 45 48 47 PRTEscherichia coli 48 Leu Asp Asn Val Ala Ala Lys Ala Gly Tyr Ser Lys TrpHis Leu Gln 1 5 10 15 Arg Met Phe Lys Asp Val Thr Gly His Ala Ile GlyAla Tyr Ile Arg 20 25 30 Ala Arg Arg Leu Ser Lys Ser Ala Val Ala Leu ArgLeu Thr Ala 35 40 45 49 47 PRT Escherichia coli 49 Leu Asp Asp Val AlaAsn Lys Ala Gly Tyr Thr Lys Trp Tyr Phe Gln 1 5 10 15 Arg Leu Phe LysLys Val Thr Gly Val Thr Leu Ala Ser Tyr Ile Arg 20 25 30 Ala Arg Arg LeuThr Lys Ala Ala Val Glu Leu Arg Leu Thr Lys 35 40 45 50 47 PRTSalmonella typhimurium 50 Ile Asp Val Val Ala Lys Lys Ser Gly Tyr SerLys Trp Tyr Leu Gln 1 5 10 15 Arg Met Phe Arg Thr Val Thr His Gln ThrLeu Gly Glu Tyr Ile Arg 20 25 30 Gln Arg Arg Leu Leu Leu Ala Ala Val GluLeu Arg Thr Thr Glu 35 40 45 51 47 PRT Escherichia coli 51 Ile Asp ValVal Ala Lys Lys Ser Gly Tyr Ser Lys Trp Tyr Leu Gln 1 5 10 15 Arg MetPhe Arg Thr Val Thr His Gln Thr Leu Gly Asp Tyr Ile Arg 20 25 30 Gln ArgArg Leu Leu Leu Ala Ala Val Glu Leu Arg Thr Thr Glu 35 40 45 52 47 PRTEscherichia coli 52 Ile Glu Asp Ile Ala Gln Lys Ser Gly Tyr Ser Arg ArgAsn Ile Gln 1 5 10 15 Leu Leu Phe Arg Asn Phe Met His Val Pro Leu GlyGlu Tyr Ile Arg 20 25 30 Lys Arg Arg Leu Cys Arg Ala Ala Ile Leu Val ArgLeu Thr Ala 35 40 45 53 47 PRT Yersinia pestis 53 Ile Asp Cys Leu ValLeu Tyr Ser Gly Phe Ser Arg Arg Tyr Leu Gln 1 5 10 15 Ile Ser Phe LysGlu Tyr Val Gly Met Pro Ile Gly Thr Tyr Ile Arg 20 25 30 Val Arg Arg AlaSer Arg Ala Ala Ala Leu Leu Arg Leu Thr Arg 35 40 45 54 47 PRTEnterobacter freundii 54 Ile Glu Asp Ile Ala Arg His Ala Gly Tyr Ser LysTrp His Leu Gln 1 5 10 15 Arg Leu Phe Leu Gln Tyr Lys Gly Glu Ser LeuGly Arg Tyr Ile Arg 20 25 30 Glu Arg Lys Leu Leu Leu Ala Ala Arg Asp LeuArg Glu Ser Asp 35 40 45 55 47 PRT Klebsiella pneumoniae 55 Ile Asp AspIle Ala Arg His Ala Gly Tyr Ser Lys Trp His Leu Gln 1 5 10 15 Arg LeuPhe Leu Gln Tyr Lys Gly Glu Ser Leu Gly Arg Tyr Ile Arg 20 25 30 Glu ArgLys Leu Leu Leu Ala Ala Arg Asp Leu Arg Asp Thr Asp 35 40 45 56 48 PRTRhizobium species 56 Ile Glu Asp Leu Ala Ala Ala Ala Arg Cys Thr Pro ArgAla Leu Gln 1 5 10 15 Arg Met Phe Arg Thr Tyr Arg Gly Gly Ser Pro MetSer Val Leu Cys 20 25 30 Asn Tyr Arg Leu Ala Ala Ala His Gly Ala Ile LysAla Gly Arg Ala 35 40 45 57 50 PRT Ralstonia solanacearum 57 Thr Arg GluVal Ala Ala His Ile Asn Val Thr Glu Arg Ala Leu Gln 1 5 10 15 Leu AlaPhe Lys Ser Ala Val Gly Met Ser Pro Ser Ser Val Ile Arg 20 25 30 Arg MetArg Leu Glu Gly Ile Arg Ser Asp Leu Leu Asp Ser Glu Arg 35 40 45 Asn Pro50 58 50 PRT Rhodococcus erythropolis 58 Val Ala Gln Val Ala Arg Asn ValGly Val Ser Val Arg Ser Leu Gln 1 5 10 15 Val Gly Phe Gln Asn Ser LeuGly Thr Thr Pro Met Arg Gln Leu Lys 20 25 30 Ile Arg Ile Met Gln Lys AlaArg Lys Asp Leu Leu Arg Ala Asp Pro 35 40 45 Ala Ser 50 59 50 PRTPseudomonas putidas 59 Leu Glu Arg Leu Ala Glu Leu Ala Met Met Ser ProArg Ser Leu Tyr 1 5 10 15 Asn Leu Phe Glu Lys His Ala Gly Thr Thr ProLys Asn Tyr Ile Arg 20 25 30 Asn Arg Lys Leu Glu Ser Ile Arg Ala Cys LeuAsn Asp Pro Ser Ala 35 40 45 Asn Val 50 60 50 PRT Pseudomonas putidas 60Leu Glu Arg Leu Ala Glu Leu Ala Met Met Ser Pro Arg Ser Leu Tyr 1 5 1015 Asn Leu Phe Glu Lys His Ala Gly Thr Thr Pro Lys Asn Tyr Ile Arg 20 2530 Asn Arg Lys Leu Glu Ser Ile Arg Ala Cys Leu Asn Asp Pro Ser Ala 35 4045 Asn Val 50 61 50 PRT Pseudomonas putidas 61 Leu Glu Arg Leu Ala GluLeu Ala Met Met Ser Pro Arg Ser Leu Tyr 1 5 10 15 Asn Leu Phe Glu LysHis Ala Gly Thr Thr Pro Lys Asn Tyr Ile Arg 20 25 30 Asn Arg Lys Leu GluCys Ile Arg Ala Arg Leu Ser Asp Pro Asn Ala 35 40 45 Asn Val 50 62 50PRT Pseudomonas putidas 62 Leu Glu Arg Leu Ala Glu Leu Ala Met Met SerPro Arg Ser Leu Tyr 1 5 10 15 Asn Leu Phe Glu Lys His Ala Gly Thr ThrPro Lys Asn Tyr Ile Arg 20 25 30 Asn Arg Lys Leu Glu Cys Ile Arg Ala ArgLeu Ser Asp Pro Asn Ala 35 40 45 Asn Val 50 63 50 PRT Pseudomonasputidas 63 Leu Glu Gln Leu Ala Glu Leu Ala Leu Met Ser Pro Arg Ser LeuTyr 1 5 10 15 Thr Met Phe Glu Lys His Thr Gly Thr Thr Pro Met Asn TyrIle Arg 20 25 30 Asn Arg Lys Leu Glu Cys Val Arg Ala Cys Leu Ser Asn ProThr Thr 35 40 45 Asn Tyr 50 64 50 PRT Escherichia coli 64 Val Leu AspLeu Cys Asn Gln Leu His Val Ser Arg Arg Thr Leu Gln 1 5 10 15 Asn ArgPhe His Ala Ile Leu Gly Ile Gly Arg Asn Ala Trp Leu Lys 20 25 30 Arg IleArg Leu Asn Ala Val Arg Arg Glu Leu Ile Ser Pro Trp Ser 35 40 45 Gln Ser50 65 43 PRT Mycobacterium tuberculosis 65 Ile Ala Asp Gln Leu Asp MetHis Pro Arg Thr Leu Gln Arg Arg Leu 1 5 10 15 Ala Ala Glu Gly Leu ArgCys His Asp Leu Ile Glu Arg Glu Arg Arg 20 25 30 Ala Gln Ala Ala Arg TyrLeu Ala Gln Pro Gly 35 40 66 43 PRT Escherichia coli 66 Val Ala Arg TyrLeu Tyr Ile Ser Val Ser Thr Leu His Arg Arg Leu 1 5 10 15 Ala Ser GluGly Val Ser Phe Gln Phe Ile Leu Asp Asp Val Arg Leu 20 25 30 Asn Asn AlaLeu Ser Ala Ile Gln Thr Thr Val 35 40 67 46 PRT Escherichia coli 67 LeuSer Met Val Ala Ser Cys Leu Cys Leu Ser Pro Ser Leu Leu Lys 1 5 10 15Lys Lys Leu Lys Ser Glu Asn Thr Ser Tyr Ser Gln Ile Ile Thr Thr 20 25 30Cys Arg Met Arg Tyr Ala Val Asn Glu Leu Met Met Asp Gly 35 40 45 68 46PRT Escherichia coli 68 Leu Arg Ile Val Ala Ser Ser Leu Cys Leu Ser ProSer Leu Leu Lys 1 5 10 15 Lys Lys Leu Lys Asn Glu Asn Thr Ser Tyr SerGln Ile Val Thr Glu 20 25 30 Cys Arg Met Arg Tyr Ala Val Gln Met Leu LeuMet Asp Asn 35 40 45 69 46 PRT Escherichia coli 69 Leu Ala Arg Ile AlaSer Glu Leu Leu Met Ser Pro Ser Leu Leu Lys 1 5 10 15 Lys Lys Leu ArgGlu Glu Glu Thr Ser Tyr Ser Gln Leu Leu Thr Glu 20 25 30 Cys Arg Met GlnArg Ala Leu Gln Leu Ile Val Ile His Gly 35 40 45 70 46 PRT Escherichiacoli 70 Leu Lys Asp Ile Ala Glu Leu Ile Tyr Thr Ser Glu Ser Leu Ile Lys1 5 10 15 Lys Arg Leu Arg Asp Glu Gly Thr Ser Phe Thr Glu Ile Leu ArgAsp 20 25 30 Thr Arg Met Arg Tyr Ala Lys Lys Leu Ile Thr Ser Asn Ser 3540 45 71 46 PRT Escherichia coli 71 Leu Arg Asp Ile Ala Glu Arg Met TyrThr Ser Glu Ser Leu Ile Lys 1 5 10 15 Lys Lys Leu Gln Asp Glu Asn ThrCys Phe Ser Lys Ile Leu Leu Ala 20 25 30 Ser Arg Met Ser Met Ala Arg ArgLeu Leu Glu Leu Arg Gln 35 40 45 72 46 PRT Escherichia coli 72 Leu AspAsp Val Ala Lys Ala Leu Phe Thr Thr Pro Ser Thr Leu Arg 1 5 10 15 ArgHis Leu Asn Arg Glu Gly Val Ser Phe Arg Gln Leu Leu Leu Asp 20 25 30 ValArg Met Gly Met Ala Leu Asn Tyr Leu Thr Phe Ser Asn 35 40 45 73 46 PRTProteus inconstans 73 Leu Asp Asp Val Ala Lys Ala Leu Tyr Thr Thr ProSer Thr Leu Arg 1 5 10 15 Arg His Leu Asn Lys Glu Gly Val Ser Phe CysGln Leu Leu Leu Asp 20 25 30 Val Arg Ile Pro Ile Ala Leu Asn Tyr Leu ThrPhe Ser Asn 35 40 45 74 46 PRT Escherichia coli 74 Leu Ala Ile Ile AlaAsp Glu Phe Asn Val Ser Glu Ile Thr Ile Arg 1 5 10 15 Lys Arg Leu GluSer Glu Tyr Ile Thr Phe Asn Gln Ile Leu Met Gln 20 25 30 Ser Arg Met SerLys Ala Ala Leu Leu Leu Leu Asp Asn Ser 35 40 45 75 46 PRT Escherichiacoli 75 Leu Gly Ile Ile Ala Asp Asp Ala Asn Ala Ser Glu Ile Thr Ile Arg1 5 10 15 Lys Arg Leu Glu Ser Glu Tyr Ile Thr Phe Asn Gln Ile Leu MetGln 20 25 30 Ser Arg Met Ser Lys Ala Ala Leu Leu Leu Leu Asp Asn Ser 3540 45 76 46 PRT Escherichia coli 76 Leu Gly Ile Ile Ala Asp Asp Val ValAla Ser Glu Ile Thr Ile Arg 1 5 10 15 Lys Arg Leu Glu Ser Glu Tyr IleThr Phe Asn Gln Ile Leu Met Gln 20 25 30 Ser Arg Met Ser Lys Ala Ala LeuLeu Leu Leu Asp Asn Ser 35 40 45 77 46 PRT Escherichia coli 77 Leu AlaIle Ile Ala Asp Val Phe Asn Val Ser Glu Ile Thr Ile Arg 1 5 10 15 LysArg Leu Glu Ser Glu Asp Thr Asn Phe Asn Gln Ile Leu Met Gln 20 25 30 SerArg Met Ser Lys Ala Ala Leu Leu Leu Leu Glu Asn Ser 35 40 45 78 46 PRTEscherichia coli 78 Leu Ser Asp Ile Ala Glu Glu Met His Ile Ser Glu IleSer Val Arg 1 5 10 15 Lys Arg Leu Glu Gln Glu Cys Leu Asn Phe Asn GlnLeu Ile Leu Asp 20 25 30 Val Arg Met Asn Gln Ala Ala Lys Phe Ile Ile ArgSer Asp 35 40 45 79 46 PRT Shigella dysneteriae 79 Leu Ser Asp Ile SerAsn Asn Leu Asn Leu Ser Glu Ile Ala Val Arg 1 5 10 15 Lys Arg Leu GluSer Glu Lys Leu Thr Phe Gln Gln Ile Leu Leu Asp 20 25 30 Ile Arg Met HisHis Ala Ala Lys Leu Leu Leu Asn Ser Gln 35 40 45 80 46 PRT Escherichiacoli 80 Leu Gly Asp Val Ser Ser Ser Met Phe Met Ser Asp Ser Cys Leu Arg1 5 10 15 Lys Gln Leu Asn Lys Glu Asn Leu Thr Phe Lys Lys Ile Met LeuAsp 20 25 30 Ile Lys Met Lys His Ala Ser Leu Phe Leu Arg Thr Thr Asp 3540 45 81 46 PRT Vibrio cholera 81 Trp Ala Asp Ile Cys Gly Glu Leu ArgThr Asn Arg Met Ile Leu Lys 1 5 10 15 Lys Glu Leu Glu Ser Arg Gly ValLys Phe Arg Glu Leu Ile Asn Ser 20 25 30 Ile Arg Ile Ser Tyr Ser Ile SerLeu Met Lys Thr Gly Glu 35 40 45 82 45 PRT Escherichia coli 82 Ile AlaGly Glu Thr Gly Met Ser Val Arg Ser Leu Tyr Arg Met Phe 1 5 10 15 AlaAsp Lys Gly Leu Val Val Ala Gln Tyr Ile Arg Asn Arg Arg Leu 20 25 30 AspPhe Cys Ala Asp Ala Ile Arg His Ala Ala Asp Asp 35 40 45 83 40 PRTBacillus subtilis 83 Ala Leu His Tyr His Gln Asp Tyr Val Ser Arg Cys MetGln Gln Val 1 5 10 15 Leu Gly Val Thr Pro Ala Gln Tyr Thr Asn Arg ValArg Met Thr Glu 20 25 30 Ala Lys Arg Leu Ser Ser Thr Asn 35 40 84 8 PRTArtificial Sequence consensus sequence 84 Ala Ser Leu Phe Gly Arg AlaLeu 1 5 85 31 PRT Echerichia coli 85 Glu Pro Ile Leu Tyr Leu Ala Glu ArgTyr Gly Phe Glu Ser Gln Gln 1 5 10 15 Thr Leu Thr Arg Thr Phe Lys AsnTyr Phe Asp Val Pro Pro His 20 25 30 86 31 PRT Salmonella typhimurium 86Leu Met Ile Ser Glu Ile Ser Met Gln Cys Gly Phe Glu Asp Ser Asn 1 5 1015 Tyr Phe Ser Val Val Phe Thr Arg Glu Thr Gly Met Thr Pro Ser 20 25 3087 31 PRT Escherichia coli 87 Leu Leu Ile Ser Asp Ile Ser Thr Glu CysGly Phe Glu Asp Ser Asn 1 5 10 15 Tyr Phe Ser Asx Val Phe Thr Arg GluThr Gly Met Thr Pro Ser 20 25 30 88 31 PRT Escherichia coli 88 Ala SerVal Thr Asp Ile Ala Tyr Arg Cys Gly Phe Ser Asp Ser Asn 1 5 10 15 HisPhe Ser Thr Leu Phe Arg Arg Glu Phe Asn Trp Ser Pro Arg 20 25 30 89 31PRT Salmonella typhimurium 89 His Ser Val Thr Glu Ile Ala Tyr Arg CysGly Phe Gly Asp Ser Asn 1 5 10 15 His Phe Ser Thr Leu Phe Arg Arg GluPhe Asn Trp Ser Pro Arg 20 25 30 90 31 PRT Pseudomonas aeroginosa 90 MetSer Ile Val Asp Ile Ala Met Glu Ala Gly Phe Ser Ser Gln Ser 1 5 10 15Tyr Phe Thr Gln Ser Tyr Arg Arg Arg Phe Gly Cys Thr Pro Ser 20 25 30 9131 PRT Yersinia pestis 91 Met Ser Ile Val Asp Ile Ala Met Glu Ala GlyPhe Ser Ser Gln Ser 1 5 10 15 Tyr Phe Thr Gln Ser Tyr Arg Arg Arg PheGly Cys Thr Pro Ser 20 25 30 92 31 PRT Yersinia enterocolitica 92 MetSer Ile Val Asp Ile Ala Met Glu Ala Gly Phe Ser Ser Gln Ser 1 5 10 15Tyr Phe Thr Gln Ser Tyr Arg Arg Arg Phe Gly Cys Thr Pro Ser 20 25 30 9331 PRT Citrobacter freundii 93 Met Pro Ile Ala Thr Val Gly Arg Asn ValGly Phe Asp Asp Gln Leu 1 5 10 15 Tyr Phe Ser Arg Val Phe Lys Lys CysThr Gly Ala Ser Pro Ser 20 25 30 94 31 PRT Escherichia coli 94 Met ProIle Ala Thr Val Gly Arg Asn Val Gly Phe Asp Asp Gln Leu 1 5 10 15 TyrPhe Ser Arg Val Phe Lys Lys Cys Thr Gly Ala Ser Pro Ser 20 25 30 95 31PRT Salmonella typhimurium 95 Met Pro Ile Ala Thr Val Gly Arg Asn ValGly Phe Asp Asp Gln Leu 1 5 10 15 Tyr Phe Ser Arg Val Phe Lys Lys CysThr Gly Ala Ser Pro Ser 20 25 30 96 31 PRT Erwina chrysanthemi 96 GluSer Ile Ala Asn Ile Gly Arg Val Val Gly Tyr Asp Asp Gln Leu 1 5 10 15Tyr Phe Ser Arg Val Phe Arg Lys Arg Val Gly Val Ser Pro Ser 20 25 30 9731 PRT Salmonella typhimurium 97 Trp Ser Ile Ala Ser Ile Ala Arg Asn LeuGly Phe Ser Gln Thr Ser 1 5 10 15 Tyr Phe Cys Lys Val Phe Arg Gln ThrTyr Gln Val Thr Pro Gln 20 25 30 98 31 PRT Escherichia coli 98 Leu LysVal Lys Glu Val Ala His Ala Cys Gly Phe Val Asp Ser Asn 1 5 10 15 TyrPhe Cys Arg Leu Phe Arg Lys Asn Thr Glu Arg Ser Pro Ser 20 25 30 99 31PRT Bacillus subtilis 99 Cys Lys Leu Lys Glu Ile Ala His Gln Thr Gly TyrGln Asp Glu Phe 1 5 10 15 Tyr Phe Ser Arg Ile Phe Lys Lys Tyr Thr GlyCys Ser Pro Thr 20 25 30 100 30 PRT Bacillus subtilis 100 Leu Ser IleLys Glu Ile Ala Glu Glu Ile Gly Phe Ser Val His Tyr 1 5 10 15 Phe ThrArg Val Phe Ser Ala Lys Ile Gly Ser Ser Pro Gly 20 25 30 101 30 PRTPhotobacterium leiognathi 101 Lys Pro Leu Ser Gln Val Ala Gln Leu CysGly Phe Ser Ser Gln Ser 1 5 10 15 Ser Phe Ser Gln Ala Phe Arg Arg LeuTyr Gly Met Ser Pro 20 25 30 102 31 PRT Streptomyces atratus 102 Ala ProLeu Ala Ala Ile Ala His Ser Val Gly Tyr Gly Ser Glu Ser 1 5 10 15 AlaLeu Ser Val Ala Phe Lys Arg Val Leu Gly Met Asn Pro Gly 20 25 30 103 31PRT Streptomyces limosus 103 Ala Thr Leu Ala Ser Ile Ala His Ser Val GlyTyr Gly Ser Glu Ser 1 5 10 15 Ala Leu Ser Val Ala Phe Lys Arg Val LeuGly Met Pro Pro Gly 20 25 30 104 31 PRT Escherichia coli 104 Leu Pro ValVal Val Ile Ala Glu Ser Val Gly Tyr Ala Ser Glu Ser 1 5 10 15 Ser PheHis Lys Ala Phe Val Arg Glu Phe Gly Cys Thr Pro Gly 20 25 30 105 31 PRTHaemophilus influenzae 105 Gln Ser Val Leu Ala Ile Ala Leu Glu Val GlyTyr Gln Ser Glu Ala 1 5 10 15 His Phe Cys Lys Val Phe Lys Asn Tyr TyrGln Leu Ser Pro Ser 20 25 30 106 31 PRT Pseudomonas aeroginosa 106 GlnSer Val Ala Arg Val Gly Gln Ala Val Gly Tyr Asp Asp Ser Tyr 1 5 10 15Tyr Phe Ser Arg Leu Phe Ser Lys Val Met Gly Leu Ser Pro Ser 20 25 30 10731 PRT Streptococcus mutans 107 Leu Ser Ile Ala Glu Ile Ser Asn Ser ValGly Phe Ser Asp Ser Leu 1 5 10 15 Ala Phe Ser Lys Ala Phe Lys Asn TyrPhe Gly Lys Ser Pro Ser 20 25 30 108 31 PRT Pediococcus pentosaceus 108Asn Ser Val Gln Ser Ile Ala Asn Met Tyr Gly Tyr Lys Asp Ser Phe 1 5 1015 Thr Phe Ser Lys Ala Phe Lys Arg Tyr Ser Gly Ala Ser Pro Ser 20 25 30109 31 PRT Escherichia coli 109 Asn Ser Val Gly Glu Val Ala Asp Thr LeuAsn Phe Phe Asp Ser Phe 1 5 10 15 His Phe Ser Lys Ala Phe Lys His LysPhe Gly Tyr Ala Pro Ser 20 25 30 110 30 PRT Pseudoalteromonascarragenorora 110 Ser Val Thr Glu Thr Ala Tyr Glu Val Gly Phe Asn AsnSer Asn Tyr 1 5 10 15 Phe Ala Thr Val Phe Lys Lys Arg Thr Asn Tyr ThrPro Lys 20 25 30 111 31 PRT Escherichia coli 111 Leu Ser Ile Asn Glu IleSer Gln Met Cys Gly Tyr Pro Ser Leu Gln 1 5 10 15 Tyr Phe Tyr Ser ValPhe Lys Lys Ala Tyr Asp Thr Thr Pro Lys 20 25 30 112 31 PRT Haemophilusinfluenzae 112 Ile Ser Ile Lys Glu Ile Thr Glu Ile Cys Gly Tyr Pro SerIle Gln 1 5 10 15 Tyr Phe Tyr Ser Val Phe Lys Lys Glu Phe Glu Met ThrPro Lys 20 25 30 113 31 PRT Bacillus subtilis 113 Lys Ala Ile Gly AspIle Ala Ile Cys Val Gly Ile Ala Asn Ala Pro 1 5 10 15 Tyr Phe Ile ThrLeu Phe Lys Lys Lys Thr Gly Gln Thr Pro Ala 20 25 30 114 31 PRTEscherichia coli 114 Tyr Ser Val Thr Asp Ile Ala Phe Glu Ala Gly Tyr SerSer Pro Ser 1 5 10 15 Leu Phe Ile Lys Thr Phe Lys Lys Leu Thr Ser PheThr Pro Lys 20 25 30 115 31 PRT Escherichia coli 115 Lys Ser Ile Leu AspIle Ala Leu Thr Ala Gly Phe Arg Ser Ser Ser 1 5 10 15 Arg Phe Tyr SerThr Phe Gly Lys Tyr Val Gly Met Ser Pro Gln 20 25 30 116 30 PRTPseudomonas aeroginosa 116 Ala Asn Val Ser Thr Val Ala Tyr Arg Val GlyTyr Ser Pro Ala His 1 5 10 15 Phe Ser Ile Ala Phe Arg Lys Arg Tyr GlyIle Ser Pro Ser 20 25 30 117 31 PRT Salmonella typhimurium 117 Glu ProIle Leu Tyr Leu Ala Glu Arg Tyr Gly Phe Glu Ser Gln Gln 1 5 10 15 ThrLeu Thr Arg Thr Phe Lys Asn Tyr Phe Asp Val Pro Pro His 20 25 30 118 31PRT Escherichia coli 118 Glu Pro Ile Leu Tyr Leu Ala Glu Arg Tyr Gly PheGlu Ser Gln Gln 1 5 10 15 Thr Leu Thr Arg Thr Phe Lys Asn Tyr Phe AspVal Pro Pro His 20 25 30 119 31 PRT Proteus vulgaris 119 Met Ser Ile LeuAsp Ile Ala Leu Met Tyr Gly Phe Ser Ser Gln Ala 1 5 10 15 Thr Phe ThrArg Ile Phe Lys Lys His Phe Asn Thr Thr Pro Ala 20 25 30 120 31 PRTProvidencia stuartii 120 Leu Gln Val Ile Asp Ile Ala Leu Lys Tyr Gln PheAsp Ser Gln Gln 1 5 10 15 Ser Phe Ala Lys Arg Phe Lys Ala Tyr Leu GlyIle Ser Pro Ser 20 25 30 121 31 PRT Escherichia coli 121 Arg Pro Ile LeuAsp Ile Ala Leu Gln Tyr Arg Phe Asp Ser Gln Gln 1 5 10 15 Thr Phe ThrArg Ala Phe Lys Lys Gln Phe Ala Gln Thr Pro Ala 20 25 30 122 31 PRTEscherichia coli 122 Lys Thr Ile Leu Glu Ile Ala Leu Lys Tyr Gln Phe AspSer Gln Gln 1 5 10 15 Ser Phe Thr Arg Arg Phe Lys Tyr Ile Phe Lys ValThr Pro Ser 20 25 30 123 31 PRT Salmonella typhimurium 123 Arg Pro IlePhe Asp Ile Ala Met Asp Leu Gly Tyr Val Ser Gln Gln 1 5 10 15 Thr PheSer Arg Val Phe Arg Arg Glu Phe Asp Arg Thr Pro Ser 20 25 30 124 31 PRTEscherichia coli 124 Arg Pro Ile Phe Asp Ile Ala Met Asp Leu Gly Tyr ValSer Gln Gln 1 5 10 15 Thr Phe Ser Arg Val Phe Arg Arg Glu Phe Asp ArgThr Pro Ser 20 25 30 125 30 PRT Escherichia coli 125 Lys Ser Met Leu AspIle Ala Leu Ser Leu His Phe Asp Ser Gln Gln 1 5 10 15 Ser Phe Ser ArgGlu Phe Lys Lys Leu Phe Gly Cys Ser Pro 20 25 30 126 31 PRT Yersiniapestis 126 Leu Thr Ile Ile Glu Ile Ser Ala Lys Leu Phe Tyr Asp Ser GlnGln 1 5 10 15 Thr Phe Thr Arg Glu Phe Lys Lys Ile Phe Gly Tyr Thr ProArg 20 25 30 127 30 PRT Enterobacter freundii 127 Glu Arg Val Tyr GluIle Cys Leu Arg Tyr Gly Phe Glu Ser Gln Gln 1 5 10 15 Thr Phe Thr ArgIle Phe Thr Arg Thr Phe His Gln Pro Pro 20 25 30 128 15 PRT Klebsiellapneumoniae 128 Gln Arg Val Tyr Asp Ile Cys Leu Lys Tyr Gly Phe Asp SerGln 1 5 10 15 129 31 PRT Rhizobium species 129 Gly Ser Ile Thr Glu LeuAla Leu Asn Leu Gln Phe Ser Asn Pro Gly 1 5 10 15 Arg Phe Ser Val LeuTyr Lys Ser Ala Tyr Gly Leu Ser Pro Ser 20 25 30 130 31 PRT Bacillussubtilis 130 Ser Asn Ile Ile Asp Thr Ala Ser Arg Trp Gly Ile Arg Ser ArgSer 1 5 10 15 Ala Leu Val Lys Gly Tyr Arg Lys Gln Phe Asn Glu Ala ProSer 20 25 30 131 31 PRT Rhodococcus erythropolis 131 Glu Gly Val Thr GluIle Ala Gln Arg Trp Gly Phe Leu His Val Gly 1 5 10 15 Arg Phe Ala GlyGlu Tyr Lys Gln Thr Phe Gly Val Ser Pro Ser 20 25 30 132 31 PRTPseudomonas putidas 132 Arg Ser Ile Thr Glu Ile Ala Leu Asp Tyr Gly PheLeu His Leu Gly 1 5 10 15 Arg Phe Ala Glu Asn Tyr Arg Ser Ala Phe GlyGlu Leu Pro Ser 20 25 30 133 31 PRT Pseudomonas putidas 133 Arg Ser IleThr Glu Ile Ala Leu Asp Tyr Gly Phe Leu His Leu Gly 1 5 10 15 Arg PheAla Glu Asn Tyr Arg Ser Ala Phe Gly Glu Leu Pro Ser 20 25 30 134 31 PRTPseudomonas putidas 134 Arg Ser Val Thr Glu Met Ala Leu Asp Tyr Gly PhePhe His Thr Gly 1 5 10 15 Arg Phe Ala Glu Asn Tyr Arg Ser Thr Phe GlyGlu Leu Pro Ser 20 25 30 135 31 PRT Escherichia coli 135 Arg Ser Val ThrGlu Met Ala Leu Asp Tyr Gly Phe Phe His Thr Gly 1 5 10 15 Arg Phe AlaGlu Asn Tyr Arg Ser Thr Phe Gly Glu Leu Pro Ser 20 25 30 136 31 PRTMycobacterium tuberculosis 136 Arg Ser Ile Thr Glu Val Ala Leu Asp TyrGly Phe Leu His Leu Gly 1 5 10 15 Arg Phe Ala Glu Lys Tyr Arg Ser ThrPhe Gly Glu Leu Pro Ser 20 25 30 137 31 PRT Escherichia coli 137 Met ThrVal Lys Asp Ala Ala Met Gln Trp Gly Phe Trp His Leu Gly 1 5 10 15 GlnPhe Ala Thr Asp Tyr Gln Gln Leu Phe Ser Glu Lys Pro Ser 20 25 30 138 31PRT Escherichia coli 138 Leu Tyr Leu Ser Gln Ile Ala Val Leu Leu Gly TyrSer Glu Gln Ser 1 5 10 15 Ala Arg Asn Arg Ser Cys Arg Arg Trp Phe GlyMet Thr Pro Arg 20 25 30 139 30 PRT Escherichia coli 139 Lys Pro Ile SerGlu Ile Ala Arg Glu Asn Gly Tyr Lys Cys Pro Ser 1 5 10 15 Arg Phe ThrGlu Arg Phe His Asn Arg Phe Asn Ile Thr Pro 20 25 30 140 31 PRTEscherichia coli 140 Lys Asn Ile Ser Gln Val Ser Gln Ser Cys Gly Tyr AsnSer Thr Ser 1 5 10 15 Tyr Phe Ile Ser Val Phe Lys Asp Phe Tyr Gly MetThr Pro Leu 20 25 30 141 31 PRT Escherichia coli 141 Lys Asn Ile Thr GlnVal Ala Gln Leu Cys Gly Tyr Ser Ser Thr Ser 1 5 10 15 Tyr Phe Ile SerVal Phe Lys Ala Phe Tyr Gly Leu Thr Pro Leu 20 25 30 142 31 PRTEscherichia coli 142 Phe Ser Ile Lys Arg Val Ala Val Ser Cys Gly Tyr HisSer Val Ser 1 5 10 15 Tyr Phe Ile Tyr Val Phe Arg Asn Tyr Tyr Gly MetThr Pro Thr 20 25 30 143 31 PRT Escherichia coli 143 Tyr Ser Ile Asn ValVal Ala Gln Lys Cys Gly Tyr Asn Ser Thr Ser 1 5 10 15 Tyr Phe Ile CysAla Phe Lys Asp Tyr Tyr Gly Val Thr Pro Ser 20 25 30 144 31 PRT Proteusinconstans 144 Ile Pro Leu His Thr Ile Ala Glu Lys Cys Gly Tyr Ser SerThr Ser 1 5 10 15 Tyr Phe Ile Asn Thr Phe Arg Gln Tyr Tyr Gly Val ThrPro His 20 25 30 145 31 PRT Escherichia coli 145 Tyr Ser Val Phe Gln IleSer His Arg Cys Gly Phe Gly Ser Asn Ala 1 5 10 15 Tyr Phe Cys Asp ValPhe Lys Arg Lys Tyr Asn Met Thr Pro Ser 20 25 30 146 31 PRT Escherichiacoli 146 Tyr Ser Val Phe Gln Ile Ser His Arg Cys Gly Phe Gly Ser Asn Ala1 5 10 15 Tyr Phe Cys Asp Ala Phe Lys Arg Lys Tyr Gly Met Thr Pro Ser 2025 30 147 31 PRT Escherichia coli 147 Tyr Gln Ile Ser Gln Ile Ser AsnMet Ile Gly Phe Ser Ser Thr Ser 1 5 10 15 Tyr Phe Ile Arg Leu Phe ValLys His Phe Gly Ile Thr Pro Lys 20 25 30 148 31 PRT Escherichia coli 148Tyr Gln Ile Ser Gln Ile Ser Asn Met Ile Gly Ile Ser Ser Ala Ser 1 5 1015 Tyr Phe Ile Arg Val Phe Asn Lys His Tyr Gly Val Thr Pro Lys 20 25 30149 31 PRT Escherichia coli 149 Tyr Gln Ile Ser Gln Ile Ser Asn Met IleGly Ile Ser Ser Ala Ser 1 5 10 15 Tyr Phe Ile Arg Ile Phe Asn Lys HisTyr Gly Val Thr Pro Lys 20 25 30 150 31 PRT Escherichia coli 150 Tyr GlnIle Ser Gln Ile Ser Asn Met Ile Gly Ile Ser Ser Ala Ser 1 5 10 15 TyrPhe Ile Arg Ile Phe Asn Lys His Phe Gly Val Thr Arg Ser 20 25 30 151 31PRT Escherichia coli 151 His Gln Ile Gly Met Ile Ala Ser Leu Val Gly TyrThr Ser Val Ser 1 5 10 15 Tyr Phe Ile Lys Thr Phe Lys Glu Tyr Tyr GlyVal Thr Pro Lys 20 25 30 152 31 PRT Escherichia coli 152 Ser Tyr Ile AsnAsp Val Ser Arg Leu Ile Gly Ile Ser Ser Pro Ser 1 5 10 15 Tyr Phe IleArg Lys Phe Asn Glu Tyr Tyr Gly Ile Thr Pro Lys 20 25 30 153 31 PRTVibrio cholera 153 Lys Asn Ile Asp Glu Ile Ser Cys Leu Val Gly Phe AsnSer Thr Ser 1 5 10 15 Tyr Phe Ile Lys Val Phe Lys Glu Tyr Tyr Asn ThrThr Pro Lys 20 25 30 154 31 PRT Vibrio cholera 154 Phe Lys Ile Lys GlnIle Ala Tyr Gln Ser Gly Phe Ala Ser Val Ser 1 5 10 15 Asn Phe Ser ThrVal Phe Lys Ser Thr Met Asn Val Ala Pro Ser 20 25 30 155 30 PRTEscherichia coli 155 Glu Lys Leu Ala Gly Ile Gly Phe His Trp Gly Phe SerAsp Gln Ser 1 5 10 15 His Phe Ser Thr Val Phe Lys Gln Arg Phe Gly MetThr Pro 20 25 30 156 30 PRT Bacillus subtilis 156 Asp Lys Met Gly ValIle Ala Glu Thr Val Gly Met Glu Asp Pro Thr 1 5 10 15 Tyr Phe Ser LysLeu Phe Lys Gln Ile Glu Gly Ile Ser Pro 20 25 30 157 10 PRT ArtificialSequence consensus sequence 157 Ile Ile Ala Gly Phe Ser Phe Lys Gly Pro1 5 10 158 45 PRT Escherichia coli 158 Leu Ser Leu Glu Lys Val Ser GluArg Ser Gly Tyr Ser Lys Trp His 1 5 10 15 Leu Gln Arg Met Phe Lys LysGlu Thr Gly His Ser Leu Gly Gln Tyr 20 25 30 Ile Arg Ser Arg Lys Met ThrGlu Ile Ala Gln Lys Leu 35 40 45 159 45 PRT Providencia stuartii 159 LeuSer Leu Asp Asp Ile Ala Gln His Ser Gly Tyr Thr Lys Trp His 1 5 10 15Leu Gln Arg Val Phe Arg Lys Ile Val Gly Met Pro Leu Gly Glu Tyr 20 25 30Ile Arg Arg Arg Arg Ile Cys Glu Ala Ala Lys Glu Leu 35 40 45 160 45 PRTEscherichia coli 160 Ile Asp Ile Asn Ala Leu Val Asp Tyr Ser Gly Tyr SerArg Arg Tyr 1 5 10 15 Leu Gln Leu Leu Phe Lys Glu Asn Ile Gly Val ThrLeu Gly Lys Tyr 20 25 30 Ile Gln Leu Arg Arg Ile Thr Arg Ala Ala Ile LeuLeu 35 40 45 161 45 PRT Escherichia coli 161 Phe Asp Ile Ala Ser Val AlaGln His Val Cys Leu Ser Pro Ser Arg 1 5 10 15 Leu Ser His Leu Phe ArgGln Gln Leu Gly Ile Ser Val Leu Ser Trp 20 25 30 Arg Glu Asp Gln Arg IleSer Gln Ala Lys Leu Leu Leu 35 40 45 162 45 PRT Yersinia pestis 162 IleAsn Ile Asp Cys Leu Val Leu Tyr Ser Gly Phe Ser Arg Arg Tyr 1 5 10 15Leu Gln Ile Ser Phe Lys Glu Tyr Val Gly Met Pro Ile Gly Thr Tyr 20 25 30Ile Arg Val Arg Arg Ala Ser Arg Ala Ala Ala Leu Leu 35 40 45 163 45 PRTPhotobacterium leiognathi 163 Ile Ser Val Ala Glu Leu Ser Ser Val AlaPhe Leu Ala Gln Ser Gln 1 5 10 15 Phe Tyr Ala Leu Phe Lys Ser Gln MetGly Ile Thr Pro His Gln Tyr 20 25 30 Val Leu Arg Lys Arg Leu Asp Leu AlaLys Gln Leu Ile 35 40 45 164 45 PRT Escherichia coli 164 Leu Thr Ile AsnAsp Val Ala Glu His Val Lys Leu Asn Ala Asn Tyr 1 5 10 15 Ala Met GlyIle Phe Gln Arg Val Met Gln Leu Thr Met Lys Gln Tyr 20 25 30 Ile Thr AlaMet Arg Ile Asn His Val Arg Ala Leu Leu 35 40 45 165 45 PRT Salmonellaenterica 165 Leu Glu Leu Glu Arg Leu Ala Ala Phe Cys Asn Leu Ser Lys PheHis 1 5 10 15 Phe Val Ser Arg Tyr Lys Ala Ile Thr Gly Arg Thr Pro IleGln His 20 25 30 Phe Leu His Leu Lys Ile Glu Tyr Ala Cys Gln Leu Leu 3540 45 166 45 PRT Proteus vulgaris 166 Leu Arg Val Asn Asp Ile Ala LysLys Leu Asn Leu Ser Arg Ser Tyr 1 5 10 15 Leu Tyr Lys Ile Phe Arg LysSer Thr Asn Leu Ser Ile Lys Glu Tyr 20 25 30 Ile Leu Gln Val Arg Met LysArg Ser Gln Tyr Leu Leu 35 40 45 167 45 PRT Klebsiella pneumoniae 167Leu Ser Val Glu Gln Leu Ala Ala Glu Ala Asn Met Ser Val Ser Ala 1 5 1015 Phe His His Asn Phe Lys Ser Val Thr Ser Thr Ser Pro Leu Gln Tyr 20 2530 Leu Lys Asn Tyr Arg Leu His Lys Ala Arg Met Met Ile 35 40 45 168 45PRT Escherichia coli 168 Leu Arg Leu Glu Asp Val Ala Ser His Val Tyr LeuSer Pro Tyr Tyr 1 5 10 15 Phe Ser Lys Leu Phe Lys Lys Tyr Gln Gly IleGly Phe Asn Ala Trp 20 25 30 Val Asn Arg Gln Arg Met Val Ser Ala Arg GluLeu Leu 35 40 45 169 45 PRT Escherichia coli 169 Ile Lys Ile Asp Thr IleAla Asn Lys Ser Gly Tyr Ser Lys Trp His 1 5 10 15 Leu Gln Arg Ile PheLys Asp Phe Lys Gly Cys Thr Leu Gly Glu Tyr 20 25 30 Val Arg Lys Arg ArgLeu Leu Glu Ala Ala Lys Ser Leu 35 40 45 170 45 PRT Escherichia coli 170Leu Arg Ile Asp Asp Ile Ala Arg His Ala Gly Tyr Ser Lys Trp His 1 5 1015 Leu Gln Arg Leu Phe Leu Gln Tyr Lys Gly Glu Ser Leu Gly Arg Tyr 20 2530 Ile Arg Glu Arg Lys Leu Leu Leu Ala Ala Arg Asp Leu 35 40 45 171 45PRT Escherichia coli 171 Phe Ala Leu Asp Lys Phe Cys Asp Glu Ala Ser CysSer Glu Arg Val 1 5 10 15 Leu Arg Gln Gln Phe Arg Gln Gln Thr Gly MetThr Ile Asn Gln Tyr 20 25 30 Leu Arg Gln Val Arg Val Cys His Ala Gln TyrLeu Leu 35 40 45 172 45 PRT Escherichia coli 172 Val Asn Trp Asp Ala ValAla Asp Gln Phe Ser Leu Ser Leu Arg Thr 1 5 10 15 Leu His Arg Gln LeuLys Gln Gln Thr Gly Leu Thr Pro Gln Arg Tyr 20 25 30 Leu Asn Arg Leu ArgLeu Met Lys Ala Arg His Leu Leu 35 40 45 173 45 PRT Escherichia coli 173Leu Ser Leu Asp Asn Val Ala Ala Lys Ala Gly Tyr Ser Lys Trp His 1 5 1015 Leu Gln Arg Met Phe Lys Asp Val Thr Gly His Ala Ile Gly Ala Tyr 20 2530 Ile Arg Ala Arg Arg Leu Ser Lys Ser Ala Val Ala Leu 35 40 45 174 45PRT Escherichia coli 174 Leu Asn Ile Asp Val Val Ala Lys Lys Ser Gly TyrSer Lys Trp Tyr 1 5 10 15 Leu Gln Arg Met Phe Arg Thr Val Thr His GlnThr Leu Gly Asp Tyr 20 25 30 Ile Arg Gln Arg Arg Leu Leu Leu Ala Ala ValGlu Leu 35 40 45 175 45 PRT Escherichia coli 175 Leu Leu Leu Asp Asp ValAla Asn Lys Ala Gly Tyr Thr Lys Trp Tyr 1 5 10 15 Phe Gln Arg Leu PheLys Lys Val Thr Gly Val Thr Leu Ala Ser Tyr 20 25 30 Ile Arg Ala Arg ArgLeu Thr Lys Ala Ala Val Glu Leu 35 40 45 176 45 PRT Escherichia coli 176Ile Lys Val Asp Gln Val Leu Asp Ala Val Gly Ile Ser Arg Ser Asn 1 5 1015 Leu Glu Lys Arg Phe Lys Glu Glu Val Gly Glu Thr Ile His Ala Met 20 2530 Ile His Ala Glu Lys Leu Glu Lys Ala Arg Ser Leu Leu 35 40 45 177 45PRT Pseudomonas putidas 177 Ile Ser Leu Glu Arg Leu Ala Glu Leu Ala MetMet Ser Pro Arg Ser 1 5 10 15 Leu Tyr Asn Leu Phe Glu Lys His Ala GlyThr Thr Pro Lys Asn Tyr 20 25 30 Ile Arg Asn Arg Lys Leu Glu Ser Ile ArgAla Cys Leu 35 40 45 178 45 PRT Pseudomonas putidas 178 Ile Ser Leu GluArg Leu Ala Glu Leu Ala Leu Met Ser Pro Arg Ser 1 5 10 15 Leu Tyr ThrLeu Phe Glu Lys His Ala Gly Thr Thr Pro Lys Asn Tyr 20 25 30 Ile Arg AsnArg Lys Leu Glu Cys Ile Arg Ala Arg Leu 35 40 45 179 45 PRT Haemophilusinfluenzae 179 Trp His Ile Glu Gln Leu Ala Glu Leu Ala Thr Met Ser ArgAla Asn 1 5 10 15 Phe Ile Arg Ile Phe Gln Gln His Ile Gly Met Ser ProGly Arg Phe 20 25 30 Leu Thr Lys Val Arg Leu Gln Ser Ala Ala Phe Leu Leu35 40 45 180 45 PRT Bacillus subtilis 180 Leu Lys Leu Thr Asp Val AlaSer His Phe His Ile Ser Gly Arg His 1 5 10 15 Leu Ser Arg Leu Phe AlaAla Glu Leu Gly Val Ser Tyr Ser Glu Phe 20 25 30 Val Gln Asn Glu Lys IleAsn Lys Ala Ala Glu Leu Leu 35 40 45 181 45 PRT Bacillus subtilis 181Ile Thr Leu Ala Gln Leu Ser Gln Met Ala Gly Ile Ser Ala Lys His 1 5 1015 Tyr Ser Glu Ser Phe Lys Lys Trp Thr Gly Gln Ser Val Thr Glu Phe 20 2530 Ile Thr Lys Thr Arg Ile Thr Lys Ala Lys Arg Leu Met 35 40 45 182 22PRT Artificial Sequence consensus sequence 182 Xaa Xaa Xaa Ala Xaa XaaXaa Gly Xaa Ser Xaa Xaa Xaa Leu Gln Xaa 1 5 10 15 Xaa Phe Xaa Xaa XaaXaa 20 183 32 PRT Escherichia coli 183 Ile Leu Tyr Leu Ala Glu Arg TyrGly Phe Glu Ser Gln Gln Thr Leu 1 5 10 15 Thr Arg Thr Phe Lys Asn TyrPhe Asp Val Pro Pro His Lys Tyr Arg 20 25 30 184 32 PRT Providenciastuartii 184 Val Ile Asp Ile Ala Leu Lys Tyr Gln Phe Asp Ser Gln Gln SerPhe 1 5 10 15 Ala Lys Arg Phe Lys Ala Tyr Leu Gly Ile Ser Pro Ser LeuTyr Arg 20 25 30 185 32 PRT Escherichia coli 185 Ile Val Asp Ile Ser GluArg Leu Phe Tyr Asp Ser Gln Gln Thr Phe 1 5 10 15 Thr Arg Glu Phe LysLys Asn Ser Gly Tyr Thr Pro Leu Gln Tyr Arg 20 25 30 186 32 PRTEscherichia coli 186 Ile Ala Thr Val Gly Arg Asn Val Gly Phe Asp Asp GlnLeu Tyr Phe 1 5 10 15 Ser Arg Val Phe Lys Lys Cys Thr Gly Ala Ser ProSer Glu Phe Arg 20 25 30 187 32 PRT Yersinia pestis 187 Ile Ile Glu IleSer Ala Lys Leu Phe Tyr Asp Ser Gln Gln Thr Phe 1 5 10 15 Thr Arg GluPhe Lys Lys Ile Phe Gly Tyr Thr Pro Arg Gln Tyr Arg 20 25 30 188 32 PRTPhotobacterium leiognathi 188 Leu Ser Gln Val Ala Gln Leu Cys Gly PheSer Ser Gln Ser Ser Phe 1 5 10 15 Ser Gln Ala Phe Arg Arg Leu Tyr GlyMet Ser Pro Thr Arg Tyr Gln 20 25 30 189 32 PRT Escherichia coli 189 IleLeu Asp Ile Ala Leu Thr Ala Gly Phe Arg Ser Ser Ser Arg Phe 1 5 10 15Tyr Ser Thr Phe Gly Lys Tyr Val Gly Met Ser Pro Gln Gln Tyr Arg 20 25 30190 32 PRT Pseudomonas aeroginosa 190 Val Ala Arg Val Gly Gln Ala ValGly Tyr Asp Asp Ser Tyr Tyr Phe 1 5 10 15 Ser Arg Leu Phe Ser Lys ValMet Gly Leu Ser Pro Ser Ala Tyr Arg 20 25 30 191 32 PRT Streptococcusmutans 191 Ile Ala Glu Ile Ser Asn Ser Val Gly Phe Ser Asp Ser Leu AlaPhe 1 5 10 15 Ser Lys Ala Phe Lys Asn Tyr Phe Gly Lys Ser Pro Ser LysPhe Arg 20 25 30 192 32 PRT Escherichia coli 192 Ala Ser Ala Ala Ala MetArg Val Gly Tyr Glu Ser Ala Ser Gln Phe 1 5 10 15 Ser Arg Glu Phe LysArg Tyr Phe Gly Val Thr Pro Gly Glu Asp Ala 20 25 30 193 32 PRTSalmonella enterica 193 Ile Ala Ser Ile Ala Arg Asn Leu Gly Phe Ser GlnThr Ser Tyr Phe 1 5 10 15 Cys Lys Val Phe Arg Gln Thr Tyr Gln Val ThrPro Gln Ala Tyr Arg 20 25 30 194 32 PRT Proteus vulgaris 194 Ile Leu AspIle Ala Leu Met Tyr Gly Phe Ser Ser Gln Ala Thr Phe 1 5 10 15 Thr ArgIle Phe Lys Lys His Phe Asn Thr Thr Pro Ala Lys Phe Arg 20 25 30 195 32PRT Klebsiella pneumoniae 195 Val Tyr Asp Ile Cys Leu Lys Tyr Gly PheAsp Ser Gln Gln Thr Phe 1 5 10 15 Thr Arg Val Phe Thr Arg Thr Phe AsnGln Pro Pro Gly Ala Tyr Arg 20 25 30 196 32 PRT Escherichia coli 196 IleSer Asp Ile Ser Thr Glu Cys Gly Phe Glu Asp Ser Asn Tyr Phe 1 5 10 15Ser Val Val Phe Thr Arg Glu Thr Gly Met Thr Pro Ser Gln Trp Arg 20 25 30197 32 PRT Escherichia coli 197 Val Thr Asp Ile Ala Tyr Arg Cys Gly PheSer Asp Ser Asn His Phe 1 5 10 15 Ser Thr Leu Phe Arg Arg Glu Phe AsnTrp Ser Pro Arg Asp Ile Arg 20 25 30 198 32 PRT Escherichia coli 198 IleLeu Asp Ile Ala Leu Gln Tyr Arg Phe Asp Ser Gln Gln Thr Phe 1 5 10 15Thr Arg Ala Phe Lys Lys Gln Phe Ala Gln Thr Pro Ala Leu Tyr Arg 20 25 30199 32 PRT Escherichia coli 199 Ile Phe Asp Ile Ala Met Asp Leu Gly TyrVal Ser Gln Gln Thr Phe 1 5 10 15 Ser Arg Val Phe Arg Arg Gln Phe AspArg Thr Pro Ser Asp Tyr Arg 20 25 30 200 32 PRT Escherichia coli 200 IleLeu Glu Ile Ala Leu Lys Tyr Gln Phe Asp Ser Gln Gln Ser Phe 1 5 10 15Thr Arg Arg Phe Lys Tyr Ile Phe Lys Val Thr Pro Ser Tyr Tyr Arg 20 25 30201 32 PRT Escherichia coli 201 Ile Asn Glu Ile Ser Gln Met Cys Gly TyrPro Ser Leu Gln Tyr Phe 1 5 10 15 Tyr Ser Val Phe Lys Lys Ala Tyr AspThr Thr Pro Lys Glu Tyr Arg 20 25 30 202 32 PRT Pseudomonas putidas 202Ile Thr Glu Ile Ala Leu Asp Tyr Gly Phe Leu His Leu Gly Arg Phe 1 5 1015 Ala Glu Asn Tyr Arg Ser Ala Phe Gly Glu Leu Pro Ser Asp Thr Leu 20 2530 203 32 PRT Pseudomonas putidas 203 Val Thr Glu Met Ala Leu Asp TyrGly Phe Phe His Thr Gly Arg Phe 1 5 10 15 Ala Glu Asn Tyr Arg Ser ThrPhe Gly Glu Leu Pro Ser Asp Thr Leu 20 25 30 204 32 PRT Haemophilusinfluenzae 204 Val Leu Ala Ile Ala Leu Glu Val Gly Tyr Gln Ser Glu AlaHis Phe 1 5 10 15 Cys Lys Val Phe Lys Asn Tyr Tyr Gln Leu Ser Pro SerGln Tyr Arg 20 25 30 205 32 PRT Bacillus subtilis 205 Ser Ile Lys GluIle Ala Glu Glu Ile Gly Phe Ser Val His Tyr Phe 1 5 10 15 Thr Arg ValPhe Ser Ala Lys Ile Gly Ser Ser Pro Gly Leu Phe Arg 20 25 30 206 32 PRTBacillus subtilis 206 Leu Lys Glu Ile Ala His Gln Thr Gly Tyr Gln AspGlu Phe Tyr Phe 1 5 10 15 Ser Arg Ile Phe Lys Lys Tyr Thr Gly Cys SerPro Thr Ser Tyr Met 20 25 30 207 32 PRT Artificial Sequence consensussequence 207 Ile Xaa Asp Ile Ala Xaa Xaa Xaa Gly Phe Xaa Ser Xaa Xaa TyrPhe 1 5 10 15 Xaa Xaa Xaa Phe Xaa Xaa Xaa Xaa Gly Xaa Thr Pro Ser XaaXaa Arg 20 25 30 208 22 PRT Artificial Sequence consensus sequence 208Glu Lys Val Ser Glu Arg Ser Gly Tyr Ser Lys Trp His Leu Gln Arg 1 5 1015 Met Phe Lys Lys Glu Thr 20 209 24 PRT Artificial Sequence consensussequence 209 Ile Leu Tyr Leu Ala Glu Arg Tyr Gly Phe Glu Ser Gln Gln ThrLeu 1 5 10 15 Thr Arg Thr Phe Lys Asn Tyr Phe 20 210 24 DNA Echerichiacoli 210 gtcagagttt gttccgactc gaag 24 211 81 DNA Artificial Sequencesynthetic 211 tat ctg gca gaa cga tat ggc ttc gag tcg caa caa act ctgacc cga 48 Tyr Leu Ala Glu Arg Tyr Gly Phe Glu Ser Gln Gln Thr Leu ThrArg 1 5 10 15 acc ttc aaa aat tac ttt gat gtt ccg ccg cat 81 Thr Phe LysAsn Tyr Phe Asp Val Pro Pro His 20 25 212 24 DNA Artificial Sequencesynthetic 212 cgggtcagag cttgttgcga ctcg 24 213 24 DNA ArtificialSequence synthetic 213 gaaggttccg gtcagagttt gttg 24 214 24 DNAArtificial Sequence synthetic 214 gtaatttttc aaggttcggg tcag 24 215 48PRT Artificial Sequence consensus sequence 215 Xaa Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10 15 Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 20 25 30 Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 35 40 45 216 20 PRTArtificial Sequence consensus sequence 216 Xaa Xaa Xaa Xaa Ala Xaa XaaXaa Gly Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10 15 Xaa Xaa Xaa Xaa 20

1. A method for identifying a compound which that decreases theinfectivity or virulence of a microbe comprising: contacting apolypeptide comprising a microbial transcription factor helix-turn-helixdomain with the compound under conditions which allow interaction of thecompound with the polypeptide; and measuring the ability of the compoundto affect the activity of the microbial helix-turn-helix domain, whereinthe ability of the compound to modulate the activity of the microbialtranscription factor helix-turn-helix domain identifies the compound asone that modulates infectivity or virulence.
 2. A method for identifyinga compound that decreases the infectivity or virulence of a microbe,comprising: contacting a polyleptide comprising a Mar A familyhelix-turn-helix domain polypeptide with the compound under conditionswhich allow interaction of the compound with the polypeptide; andmeasuring the ability of the compound to affect the activity of the MarAfamily helix-turn-helix domain, wherein the ability of the compound tomodulate the activity of the MarA family helix-turn-helix domainidentifies the compound as one that modulates infectivity or virulence.3. The method of claim 2, wherein the step of measuring the ability ofthe compound to affect the activity of a MarA family helix-turn-helixdomain comprises detecting the ability of the complex to activatetranscription from a MarA family member responsive promoter.
 4. Themethod of claim 3, wherein the Mar A responsive promoter is selectedfrom the group consisting of marO, micF, and fumC
 5. The method of claim3, wherein the Mar A responsive promoter is linked to a reporter gene.6. The method of claim 5, wherein the reporter gene is selected from thegroup consisting of lacZ, luciferase, phoA, or green fluorescenceprotein.
 7. The method of claim 5, wherein the step of measuringcomprises measuring the amount of reporter gene product.
 8. The methodof claim 3, wherein the step of measuring comprises measuring the amountof RNA produced by the cell.
 9. The method of claim 3, wherein the stepof measuring comprises measuring the amount of a protein produced by thecell.
 10. The method of claim 9, wherein the step of measuring comprisesusing an antibody against a protein produced by the cell. 11-14.Cancelled.
 15. The method of claim 2, wherein said polypeptide comprisesthe helix-turn-helix domain most proximal to the carboxy terminus of theMarA family protein from which it is derived.
 16. The method of claim 2,wherein said polypeptide comprises the helix-turn-helix domain mostproximal to the amino terminus of the MarA family protein from which itis derived.
 17. The method of claim 2, wherein said polypeptide consistsof the helix-turn-helix domain most proximal to the carboxy terminus ofthe MarA family protein from which it is derived.
 18. The method ofclaim 2, wherein said polypeptide consists of the helix-turn-helixdomain most proximal to the amino terminus of the MarA family proteinfrom which it is derived.
 19. The method of claim 2, wherein the MarAfamily helix-turn-helix domain is derived from a protein selected fromthe group consisting of: MarA, RamA, AarP, Rob, SoxS, and PqrA.
 20. Themethod of claim 1 or 2, wherein the compound increases antibioticsusceptibility.
 21. Cancelled.
 22. The method of claim 1, wherein thecompound is effective against Gram negative bacteria.
 23. The method ofclaim 1, wherein the compound is effective against Gram positivebacteria.
 24. The method of claim 23, wherein the Gram positive bacteriaare from a genus selected from the group consisting of: Enterococcus,Staphylococcus, Clostridium and Streptococcus.
 25. The method of claim1, wherein the compound is effective against bacteria from the familyEnterobacteriaceae.
 26. The method of claim 1, wherein the compound iseffective against a bacteria of a genus selected from the groupconsisting of: Escherichia, Proteus, Klebsiella, Providencia,Enterobacter, Burkholderia, Pseudomonas, Aeromonas, Acinetobacter andMycobacteria. 27-47. Cancelled.
 48. The method of claim 1 or 2, whereinthe compound is derived from a library of small molecules.
 49. Themethod of claim 1 or 2, wherein the compound is a nucleic acid molecule.50. The method of claim 49, wherein the compound is an antisense orsense oligonucleitide.
 51. The method of claim 1 or 2, wherein thecompound is a naturally occurring small organic molecule. 52-54.Cancelled.
 55. The method of claim 1 or 2, wherein the polypeptide is ina cell.
 56. The method of claim 1 or 2, wherein the polypeptide is in acell-free system.
 57. A method for reducing the infectivity or virulenceof a microbe comprising: administering to a subject at risk ofdeveloping a microbial infection a compound that modulates an activityof a microbial transcription factor helix-turn-helix domain such thatthe infectivity or virulence of the microbe is reduced.
 58. The methodof claim 57, wherein the microbial transcription factor helix-turn-helixdomain is a MarA family helix-turn-helix domain.
 59. A method forreducing the infectivity or virulence of a microbe comprising:administering to a subject at risk of developing a microbial infection acompound that decreases an activity of a MarA family helix-turn-helixdomain such that the infectivity or virulence of the microbe is reduced.60. The method of claim 57 or 59, further comprising administering anantibiotic to the subject.
 61. A pharmaceutical composition for reducingthe infectivity or virulence of a microbe, comprising: a therapeuticallyeffective amount of a compound that decreases an activity of a microbialtranscription factor protein bearing a helix-turn-helix domain andreduces the infectivity or virulence of a microbe; and apharmaceutically acceptable carrier.
 62. The pharmaceutical compositionof claim 61 further comprising a second antimicrobial agent.